US 20200383331A1 IN (19United States ( 12 ) Patent Application Publication ( Pub. No.:USQO2Q/QZ8333l Al HEINRICHER (43 ) Pub . Date : Dec. 10 , 2020
( 54 ) COMPOSITIONS AND METHODS FOR AOIN 43/40 (WQOQI LARGE - SCALE IN VITRO PLANT AOIN 43/08 ( 2006.01 ) BIOCULTURE A01N 37/52 ( 2006.01 ) AOIN 4730 ( 2006.01 ) ( II ) Applicant: BQOSHIQOQT LLC , Hailey, IDUS A016 22/15 ( WQGOI ( 52 ) U.S. CI . ( 72 ) Inventor: Jackie HEINRICHER , Anacortes , WA CPC AOIN 43/90 ( 2013.01 ) ; AO1G 31/00 (US ) ( 2013.01 ) ; A01N 59/08 ( 2013.01 ) ; A01N 59/20 ( 2013.01 ) ; A01N 59/16 ( 2013.01 ) ; ( 21 ) Appl . No .: 16 /728,478 A01N 59/14 ( 2013.01 ) ; A01N 31/06 ( 2013.01 ) ; A01N 43/78 ( 2013.01 ) ; A01N ( 22 ) Filed : Dec. 27 , 2019 37/10 ( 2013.01 ) ; A01N 43/82 ( 2013.01 ) ; AOIN 59/12 ( 2013.01 ) ; AOIN 37/44 Related U.S. Application Data ( 2013.01 ) ; A01N 43/40 ( 2013.01 ) ; A01N ( 63 ) Continuation of application No. PCT /US2018 / 43/08 ( 2013.01 ) ; A01N 37/52 ( 2013.01 ) ; 040637 , filed on Jul. 2 , 2018 , Continuation of appli AOIN 47/30 ( 2013.01 ) ; A01G 22/15 cation No. PCT/ US2018 / 040646 , filed on Jul. 2 , ( 2018.02 ) ; A01N 59/00 ( 2013.01 ) 2018 . ( 60 ) Provisional application No. 62 / 527,946 , filed on Jun . ( 57 ) ABSTRACT 3Q , provisional application No. 62 /6II , & a , The present invention provides media , kits , systems , and filed on Dec. 29 , 2017 , provisional application No. methods for achieving large scale pistachio production 62 / 527,862 , filed on Jun . 30 , 2017 . within a short time via bioculture , large scale yam produc tion within a short time via bioculture, high multiplication Publication Classification rate of plants including cannabis via in vitro micropropaga ( 51 ) Int . Ci . tion , high induction rates of somatic embryos from later A01N 43/90 ( 2006.01 ) buds in bamboo , reduced production of phenolic compounds AOIN 59/00 ( 2006.01 ) in plants, high production of virus - free plants , including AOIN 59/08 ( 2006.01 ) potato , and large scale hemp production via culturing. The AOIN 59/20 ( 2006.01 ) present invention for pistachio and yam production results in AOIN 59/16 ( 2006.01 ) shorter tuber development phase and higher yield . In some AOIN 59/14 ( 2006.01 ) embodiments , the present invention provides compositions, AOIN 31/06 ( 2006.01 ) methods, and systems for the micropropagation and mass AOIN 43/78 ( 2006.01 ) production of perennials, grasses, bamboos, cannabis and AOIN 37/10 ( XQOOQI phyto - pharmaceutical plants as well as hemp plants . In some AOIN 43/82 ( 2006.01 ) embodiments , the present invention provides compositions, AOIN 59/12 ( 2006.01 ) methods , and systems for reducing the production of a AOIN 37/44 ( 2006.01 ) phenolic by a plant , such as bamboo .
200
210
12 Patent Application Publication Dec. 10 , 2020 Sheet 1 of 39 US 2020/0383331 A1
1cm
Shoottipnecrosisinpistachioplantculturedvitro(B0012Medium)
2FIG.1 Patent Application Publication Dec. 10 , 2020 Sheet 2 of 39 US 2020/0383331 A1
1cm
Newshootsdevelopedfromaxillarybudsofsingle-nodeexplantspistachioafter3days ofcultureon(B003Medium)
FIG.2 Patent Application Publication Dec. 10 , 2020 Sheet 3 of 39 US 2020/0383331 A1
Rootdevelopmentinpistachio(PistaciaatlanticaxP.intergerrina)plantsculturedonrootingmediumB0018 for2weeks.Theimageontherightisaclose-upofleftphotoarrowspointtonewroot
FIG.3 Patent Application Publication Dec. 10 , 2020 Sheet 4 of 39 US 2020/0383331 A1
3 ?
plantsgrowinginvitroonB003mediumWell-developed,healthypistachio
{
FIG.4 Patent Application Publication Dec. 10 , 2020 Sheet 5 of 39 US 2020/0383331 A1
-190 -150 Controller SecondMedia Vessel 110 Growth Vessel GasSupply
170 MediaFirst Vessel
100 130
FIG.5 Patent Application Publication Dec. 10 , 2020 Sheet 6 of 39 US 2020/0383331 A1
20
52 210 ***** go 0 **2-2 12
216 CO IKI
200
FIG.6 Patent Application Publication Dec. 10 , 2020 Sheet 7 of 39 US 2020/0383331 A1
S02 297. 232---
967 05rose
FIG.7 Patent Application Publication Dec. 10 , 2020 Sheet 8 of 39 US 2020/0383331 A1
Ht2 www.240 952 PL 2412
FIG.8 Patent Application Publication Dec.10.2020 Sheet 9 of 39 US 2020/0383331 A1
-222
22 FIG.9C REE 922
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FIG.9A @ FIG.9B
CC 2 Patent Application Publication Dec. 10 , 2020 Sheet 10 of 39 US 2020/0383331 A1
320 326 322 324
complete? Yes complete? Yes Executethirdoperatingmode ?second incubation sequence isplant propagation sequence Endplantpropagationsequence
No No
NO
Executethirdoperatingmode: complete? Yes Startplantpropagationsequence Startfirstincubationsequence Establishfluidcommunication betweenfirstmediacontainerand vesselgrowth Executefirstoperatingmode firstis incubation sequence Startsecondincubationsequence Establishfluidcommunication betweensecondmediacontainerand vesselBrowth Executesecondoperatingmode
312 302 304 306. 308 310 314 316 318
FIG.10 Patent Application Publication Dec. 10 , 2020 Sheet 11 of 39 US 2020/0383331 A1
MSorB5 BOO22 tuberization MS 1 80,000 900
MSorB5 BO021 tuberization MS 0.02 60,000 900
pre-tuberization MSorB5 tuberosumL.)tuberizationofpotato(SolanumvitropropagationandCompositionmediaforin BO019 MS 0.5 30,000 900 MurashigeandSkoogmediumsalts(1962)concentrations*Allaregivenining/l;thermolabilecoinpoundswere Gamborgetal.(1968)Vitaminmixtureasdescribedbycooledto40-50°C theautoclavedmediafilter-sterilizedandadded tuberizationpre- MSorB5 BO018 MS 1 4 30,000 900
plantpropagation MSorB50 BO017 MS" 20,000 900 5,000
Growthregulators: Carbohydrate: Bufferingagent: Solidifyingagent: Macro-andmicronutrients Ancymidol Agar Components Vitamins IAA NAA 2iP Sucrose MES 5.8pH
FIG.11 Patent Application Publication Dec. 10 , 2020 Sheet 12 of 39 US 2020/0383331 A1
MSorB5 BO027 tuberization MS 5 80.000 900
MSorB5 BOO26 tuberization MS 0.5 80,000 900
MSorB5 Compositionofadditionalmediaforinvitrotuberizationpotato(SolanumtuberosumL.) BOO25 tuberization MS 0.02 0.5 80,000 900 MurashigeandSkoogmediumsalts(1962)concentrations"Allaregiveninmg/l;thermolabilecompoundswere Gamborgetal.(1968)mixtureasdescribedby40-50CVitamin mediacooledtoaddedtheautoclavedfilter-sterilizedand tuberizationpre- MSorB5 BOO24 MS 5 30,000 900
tuberizationpre- MSorB50 BOO23 MS 1 4 0.5 30,000 900
Macro-andmicronutrients Growthregulators: Carbohydrate: Bufferingagent: Solidifyingagent: IAA 2iP Ancymidol MES Agar Component Vitamins NAA Sucrose 5.8PH
FIG.12 Patent Application Publication Dec. 10 , 2020 Sheet 13 of 39 US 2020/0383331 A1
FIG . 13
Copposed BO031 BO048 BO049 DKW BOO51 B0050 BO052 BO053
0
0
0 2
0
0 Patent Application Publication Dec. 10 , 2020 Sheet 14 of 39 US 2020/0383331 A1
MMMM
FIG.14 Patent Application Publication Dec. 10 , 2020 Sheet 15 of 39 US 2020/0383331 Al
AA/ A//// :
FIG.15 Patent Application Publication Dec. 10 , 2020 Sheet 16 of 39 US 2020/0383331 A1
FIG.16 Patent Application Publication Dec. 10 , 2020 Sheet 17 of 39 US 2020/0383331 A1
FIG.17 Patent Application Publication Dec. 10 , 2020 Sheet 18 of 39 US 2020/0383331 A1
NIINNNINAINIAI
***** WW ***
FIG.18 Patent Application Publication Dec. 10 , 2020 Sheet 19 of 39 US 2020/0383331 A1
FIG.19 FIG.20 Patent Application Publication Dec. 10 , 2020 Sheet 20 of 39 US 2020/0383331 A1
0
FIG.21 Patent Application Publication Dec. 10 , 2020 Sheet 21 of 39 US 2020/0383331 A1
FIG.22 Patent Application Publication Dec. 10 , 2020 Sheet 22 of 39 US 2020/0383331 A1
FIG.23 Patent Application Publication Dec. 10 , 2020 Sheet 23 of 39 US 2020/0383331 A1
|
FIG.24 Patent Application Publication Dec. 10 , 2020 Sheet 24 of 39 US 2020/0383331 A1
FIG.25 Patent Application Publication Dec. 10 , 2020 Sheet 25 of 39 US 2020/0383331 A1 FIG . 26A
Chemical mg/ l 80054 B0055 BOO56 BO057 BO058 BOOS9 B0060 B0061 INH.NO 1,650.00 1,650.00 1,650.00 1,650.00 1,650.00 1.650.00 1,650.00 NH .( SO ) 134,00 Ca (NO3 ) 24H20 950.00 2,500.00 950.00 950.00 950.00 950.00 950.00 950.00 MgSO4.7H , 0 370.00 250.00 370.00 370.00 370.00 370.00 370.00 370.00 MnSo..4,0 16.90 10.00 16.90 16.90 16.90 16.90 16.90 16.90 ZnSO4.7H20 8.60 2.00 8.60 8.60 8.60 8.60 8.60 8.60 C450.5H0 0.03 0.03 0,03 0.03 0.03 0.03 CaCl2240 440.00 150.00 440.00 440.00 440.00 440.00 440.00 440.00 KI 0.83 0.75 0.83 0.83 0.83 0.83 0.83 0.83 CoCl2.6H 0 0.03 0.03 0.03 0.03 0.03 HBO 6.20 6.20 6.20 6.20 6.20 6.20 6.20 Na Mo09.2H2O 0.25 0.25 0.25 0.25 0.25 0,25 0.25 0.25 K2SO4 KH PO 170.00 170.00 170.00 170.00 170.00 170.00 170.00 FeSO..7H20 27.80 27.80 27.80 27.80 27.80 27.80 27.80 27.80 Na EDTA 37.30 37.30 37.30 37.30 37.30 37.30 37.30 37.30 Myo -Inositol 100.00 100.00 Thiamine HCI 10.00 10.00 10.00 10.00 10.00 10.00 10.00 Pyridoxine HCI 1.00 1.00 1.00 Nicotinicacid 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Glycine 2.00 2.00 2.00 2.00 2.00 Ribavirine (Virazole ) 2.00 1.50 2.50 2.50 BAP 0.001 to 10,0 0,001 to 10,0 0,001 10 10.0 0.001 to 10.0 10.001 to 10.0 0.001 to 10.0 0.001 to 10.0 0.001 to 10,0 TOZ Meta - Topoline 0.01 to 5.0 0.01 to 5.0 0.01 to 5.0 0.01105.0 0.01 to 5.0 0.01 to 5.0 0.01 to 5.0 0.01t05.0 1.00 JAA 21p 3,00 3.00 Paclobutrazo GAZ Citric acid 10.00 Glutamine 100.00 Casein hydrolysate 100.00 Sucrose 30,000.00 30,000.00 3,000 to 10,000 3,000 to 10,000 30,000.00 30,000.00 Charcoal 5,000.00 1,000.00 Agar 5,000,00 5,000.00 5,000.00 5.000.00 5,000.00 5,000.00 3,000.00 3,000.00 Carregeenan 5.50 $5.50 5.50 5.50 5.50 5.50 5.80 5.80 Patent Application Publication Dec. 10 , 2020 Sheet 26 of 39 US 2020/0383331 A1
FIG . 26B
Chemical mg/ l B0062 BO063 B0064 B0065 B0066 BO067 Pulsing media 1 Pulsing mediaz NH.NO 1,650.00 1,650.00 1,650.00 1,650.00 1,650.00 1,400.00 1.650.00 1,650.00 NH (SOch Ca (NO3h 4H20 111.20 1,946.00 KNO 950.00 950.00 950.00 950.00 950.00 950.00 950.00 MgSO4.7H20 370.00 370.00 370.00 370.00 370.00 370.00 370.00 370.00 MnSO..H20 16.90 16.90 16.90 16.90 16.90 16.90 16.90 16.90 ZnSO4.7H20 8.60 8.60 8.60 8.60 8.60 8.60 8.60 8.60 CuSO4.5H 0 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 CaCl2.2H20 440.00 440.00 440.00 440.00 440.00 336.00 440.00 440.00 | XI 0.83 0.83 0.83 0.83 0.83 0.83 0.83 CoCl2.6H10 0.03 0.03 0.03 0.03 0.03 0.03 0.03 H2803 6.20 6.20 6.20 6.20 6.20 6.20 6.20 6.20 NazM004.2H20 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 K SO . 1,212.50 KH ,PO 170.00 170.00 170.00 170.00 170.00 170.00 170.00 170.00 FeSO..7H0 27.80 27.80 27.80 27.80 27.80 27.80 27.80 27.80 Na EDTA 37.30 37.30 37.30 37.30 37.30 37.30 37.30 37.30 Myo- Inositol 100.00 100.00 100.00 100.00 100.00 1,000.00 100.00 100.00 Thiamine HCI 10.00 10.00 10.00 10.00 10.00 50.00 10.00 10.00 Pyridoxine HCI 1.00 Nicotinic acid 1.00 1,00 1.00 1.00 1.00 4.00 1.00 1.00 Glycine 2.00 8.00 Ribavirine (Virazole ) 2.00 NAA 2.50 2.50 2.50 2.50 1.00 0.50 0.50 BAP 0.001 to 10.0 0.001 to 10,0 0.001 to 10.0 0.001 to 10.0 0.001 to 10,0 0.001 to 10.0 0.001 to 10.0 | 0.001 to 10.0 TDZ 0.001 to 1.0 Meta - Topoline 0.01 to 5.0 0.01 to 5.0 0.01 to 5.0 0.01 to 5.0 0.01 to 5.0 0.01 to 5.0 0.01 to 5.0 0.01105.0 ??? AA 210 3.00 3.00 3.00 3.00 3.00 Paclobutrazol 1.00 GA3 2.00 Citric acid Glutamine Casein hydrolysate Sucrose 30,000.00 30,000.00 30,000.00 30,000.00 30,000.00 30,000.00 30.000.00 30,000.00 Charcoal Agar 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 Phytagel Carregeenan 10,000.00 8,000.00 PH 5.80 5.80 5.80 5.80 5.80 5.80 5.80 5.80 Patent Application Publication Dec. 10 , 2020 Sheet 27 of 39 US 2020/0383331 A1
FIG , 27
Chemical mg / l BO068 B0069 BO070 BO071 NH4NO3 1,650.00 1,650.00 825.00 NH4 ( SO4 ) 2 134.00 KNO3 950.00 2,500.00 950.00 475.00 MgSO4.7H20 370.00 250.00 370.00 185.00 MnSO4.H20 16.90 10.00 16.90 8.45 ZnSO4.7H20 8.60 2.00 8.60 4.30 CuSO4.5H20 0.03 0.03 0.03 0.01 CaCl2.2H2O 440.00 150.00 440.00 220.00 KI 0.83 0.75 0.83 0.42 CoCl2.6H20 0.03 0.03 0.03 0.01 HBO3 6.20 3.00 6.20 3.10 Na 2 MO04.2H2O 0.25 0.25 0.25 0.13 KH2PO4 170.00 170.00 85.00 FeSO4.7H2O 27.80 27.80 27.80 13.90 Na 2EDTA 37.30 37.30 37.30 18.65 Myo - inositol 100.00 Thiamine HCI 10.00 10.00 10.00 5.00 Pyridoxine HCI 1.00 1.00 1.00 0.50 Nicotinic acid 1.00 1.00 1.00 0.50 Glycine 2.00 2.00 1.00 NAA 1.00 0.10 IBA 1.00 ??? 0.05 Glutamine 100.00 Casein hydrolysate 500.00 Sucrose 20,000.00 20,000.00 20,000.00 30,000.00 Charcoal 5,000.00 5,000.00 5,000.00 Agar 5,000.00 3,000.00 Phytagel 3,000.00 3,000.00 Carregeenan 5,000.00 PH 5,50 5.50 5.50 5.80 Patent Application Publication Dec. 10 , 2020 Sheet 28 of 39 US 2020/0383331 A1
FIG.28 Patent Application Publication Dec. 10 , 2020 Sheet 29 of 39 US 2020/0383331 A1
FIG.29 Patent Application Publication Dec. 10 , 2020 Sheet 30 of 39 US 2020/0383331 A1 BOO80 3,300.00 3,800.00 740.00 16.90 8.60 0.03 880.00 0.83 0.03 6.20 0.25 340.00 55.70 74.60 170.00 100.00 0.40 1,000.00 1,000.00 1,500.00 1,500.00 2.00 5.00 52.80 60,000.00 5,000.00 5.70 BO079 3,300.00 3,800.00 740.00 16.90 8.60 0.03 880.00 0.83 0.03 6.20 0.25 340.00 55.70 74.60 170.00 100.00 0.40 1,000.00 1,000.00 1,500.00 1,500.00 1.00 5.00 52.80 30,000.00 5,000.00 5.70 BO078 3,300.00 3,800.00 740.00 16.90 8.60 0.03 880.00 0.83 0.03 6.20 0.25 340.00 55.70 74.60 170.00 100.00 0.40 1,000.00 1,000.00 1,500.00 1,500.00 5.00 30,000.00 5,000.00 5.70 B0077 3,300.00 3,800.00 740.00 16.90 8.60 0.03 880.00 0.83 0.03 6,20 0.25 340.00 55.70 74.60 170.00 100.00 0.40 1,000.00 1,000.00 1,500.00 1,500.00 5.00 60,000.00 5,000.00 5.70 BO076 3,300.00 3,800.00 740.00 16.90 8.60 0.03 880.00 0.83 0.03 6.20 0.25 340.00 55.70 74.60 170.00 100.00 0.40 1,000.00 1,000.00 1,500.00 1,500.00 2.50 5.00 30,000.00 5,000.00 5.70 BO075 3,300.00 3,800.00 740.00 16.90 8.60 0.03 880.00 0.83 0.03 6.20 0.25 340.00 55.70 74.60 170.00 100.00 0.40 1,000.00 1,000.00 1,500.00 1,500.00 2.50 5.00 60,000.00 5,000.00 5.70 BO074 3,300.00 3,800.00 740.00 16.90 8.60 0.03 880.00 0.83 0.03 6.20 0.25 340.00 55.70 74.60 170.00 100.00 0.40 1,000.00 1,000.00 1,500.00 1,500.00 5.00 5.00 30,000.00 5,000.00 5.70 BO073 3,300.00 3,800.00 740.00 16.90 8.60 0.025 880.00 0.83 0.025 6.20 0.25 340.00 55.70 74.60 170.00 100.00 0.40 1,000.00 1,000.00 1,500.00 1,500.00 2.50 5.00 30,000.00 5,000.00 5.70 BO072 1,650.00 1,900.00 370.00 16.90 8.60 0.025 440.00 0.83 0.025 6.20 0.25 170.00 55.70 74.60 170.00 100.00 0.40 1,000.00 500.00 1,500.00 1,500.00 1.00 5.00 30,000.00 5,000.00 5.70
Chemical,mg/L NHANO: NaH2PO4.H20 inositolMyo- ThiamineHCI Caseinhydrolysate KNO: ST-10 MgSO4.7H20 MnSO4.H20 ZnSO4.7H20 CuSO4.5H20 CaCl2.2H20 KI CoCl2.6H20 ???| NazM004.2H20 KH2PO4 FeSO4.7H20 NazEDTA Glutamine Proline Serine 07-200 ABA Sucrose Maltose Lactose Agar PH
FIG.30 Patent Application Publication Dec. 10 , 2020 Sheet 31 of 39 US 2020/0383331 A1
FIG . 31
Chemical , mg / L BO032 BO033 BOO34 NH4NO3 1,650.0 1,650.00 1,650.00 KNO3 1,900.0 1,900.00 1,900.00 MgSO4.7H20 370.0 370.00 370.00 MnSO4.H20 16.9 16.90 16.90 ZnSO4.7H2O 8.6 8.60 8.60 CuSO4.5H20 0.0 0.03 0.03 CaCl 2.2H20 440.0 440.00 440.00 KI 0.8 0.83 0.83 CoCl2.6H20 0.0 0.03 0.03 H3B03 6.2 6.20 6.20 Na 2M004.2H20 0.3 0.25 0.25 KH2PO4 170.0 170.00 170.00 FeSO4.7H20 55.7 55,70 55.70 Na2EDTA 74.6 74.60 74.60 Na H2PO4 H2O 170.0 170.00 170.00 Myo - inositol 100.0 100.00 100.00 Thiamine HCI 0.4 0.40 0.40 NAA 0.1 0.05 0.05 BAP 1.0 1.00 1.00 DT - 200 0.5 0.50 0.50 ST - 10 5.0 5.00 5.00 Ascorbic acid 1,000 to 10,000 Citric acid 1,000 to 10,000 Charcoal 1,000 to 5,000 Sugar 30,000.0 30,000.00 30,000.00 Agar 4,500.0 4,500.00 4,500.00 PH 5.7 5.70 5.70 Patent Application Publication Dec. 10 , 2020 Sheet 32 of 39 US 2020/0383331 A1 FIG . 32 Chemicalmo !! BOO81 BOO82 BOO83 BO084 BOO85 BOO86 B0087 BO088 BO089 80090 BO091 NH.NO 1,650.00 1,650.00 1,650.00 1,650.00 1,650.00 1,650.00 1,650.00 1,650.00 NHISOA). 134.00 134.00 134.00 0.2675 0.2675 0.2675 0.2675 1,900.00 1,900.00 1,900.00 2,500.00 1,900.00 1,900.00 1,900.00 1,900.00 1,900.00 2,500.00 2,500.00 MgSO4.7H20 370.00 370.00 370.00 250.00 370.00 370.00 370.00 370.00 370.00 250.00 250.00 16,90 16.90 16.90 10.00 16,90 16,90 16.90 16.90 16.90 10.00 ZnSO4.7H , 0 8.60 8.60 2.00 8.60 8.60 8.60 8.60 2.00 2.00 CUSO..5H20 0.025 0.025 0.025 0.029 0.025 0.025 0.025 0.025 0.025 0.025 Calla.2.0 220.00 440.00 440.00 150,00 220.00 220.00 220.00 440.00 440,00 150.00 150.00 0.83 0.83 0.83 0.75 0.83 0.83 0.83 0.83 0.75 0.75 C0026H10 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 6.20 6.20 6.20 3.00 6.20 6.20 6.20 6.20 6.20 3.00 3.00 NazMo01.2H20 0.25 0.25 0.25 0.25 0.25 0.25 0.25 170.00 170.00 170.00 85.00 85.00 85.00 170.00 170.00 FeSO..7H20 27.80 27.80 27.80 27.80 27.80 27.80 27.80 55.60 55.60 27.80 27.80 Na EDTA 37,30 37.30 37,30 37.30 37.30 37.30 37.30 74.60 74.60 37.30 37.30 D -Biotin 0.05 Folic Acid 0.50 Myo- Inositol 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Thiamine HD 10.00 10.00 10.00 10.00 10.00 0.10 10.00 Pyridoxine HD 0.50 1.00 0.50 0.50 Nicotinic acid 1.00 5.00 5.00 os 1.00 5.00 5.00 1.00 Glycine 0.50 0.50 0,50 0.50 Ribavirine Virazole) 0,05 0.02 0.10 0.02 0.02 0.50 1.00 0.50 Zeatin riboside 1,00 1.00 1.00 2,4 - D 4,00 10,000.00 2.50 Kinetine 2.00 Adenine 40.00 40.00 TOZ Meta- Topoline
JAA 1.00 2ip 2.00 Glutamine 500.00 500.00 250.00 Casein hydrolysate 1,000.00 1,000.00 100.00 Sucrose 30,000.00 2,500.00 2,500.00 30,000.00 30,000.00 30,000.00 20,000.00 30,000.00 30,000.00 20,000.00 / 30,000.00 Galactose 10,000.00 Mannitol 54,651.00 36,434,00 20,000.00 10,000.00 Agar 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 5,000.00 700.00 : 700.00 700,00 5.70 5.70 5.70 5.50 5.80 5.80 5.70 5.70 5.70 5.50 5.70 Patent Application Publication Dec. 10 , 2020 Sheet 33 of 39 US 2020/0383331 A1
YevtushenkoDmytroDrawingby
Schematicpresentationofmeristemexcisionfromyoungpotatoshootsinvitro.
wa
FIG.33 Patent Application Publication Dec. 10 , 2020 Sheet 34 of 39 US 2020/0383331 A1
medium;B0082ongrowing
FIG.34B
mediuin;BOO82ongrowing
FIG.34A Patent Application Publication Dec. 10 , 2020 Sheet 35 of 39 US 2020/0383331 A1
medium;BOO82growingon
FIG.34D AM MenstemicclonesofSolanumtuberosunov,Vazama medium;BOO82growingon meristems. istexperiment,1stdayafterisolationof
FIG.34C Patent Application Publication Dec. 10 , 2020 Sheet 36 of 39 US 2020/0383331 A1
1cm
Plantregenerationviaindirectshootorganogenesisfromthemeristemicclonesof SolanumtuberosumL.CV.Mazama,culturedonBOO83mediumArrowspoint shoots. regeneratingto
FIG.35 Patent Application Publication Dec. 10 , 2020 Sheet 37 of 39 US 2020/0383331 A1
1cm
clonesoforganogenesisfromthemeristemicPlantregenerationviaindirectshoot SolaruintuberosumLov.YukonGold,culturedonB0083mediumArrows pointto shoots.regenerating
FIG.36 Patent Application Publication Dec. 10 , 2020 Sheet 38 of 39 US 2020/0383331 A1
organogenesisthemeristemiccionesofindirectshootfromPlantregenerationvia SolariumtuberosumL.CV.RussetBurbank_2culturedonB0083mediumArrowspointto shoots.regenerating
FIG.37 Patent Application Publication Dec. 10 , 2020 Sheet 39 of 39 US 2020/0383331 A1 50016
ST-10=metatopolin FIG.38 US 2020/0383331 A1 Dec. 10 , 2020 1
COMPOSITIONS AND METHODS FOR BO011 , BO010 , BOO13 , BOO14 , BO015 , BO016 , com LARGE - SCALE IN VITRO PLANT bination thereof, or functional equivalents thereof ( e.g. , by BIOCULTURE reducing or increasing one or more component concentra tion , or by adding or removing one or more component, CROSS - REFERENCE TO RELATED wherein the media maintain the same function ). As used APPLICATIONS herein , the media named “ BOO ” is equivalent to “BOOS . ” For example , the media of the present invention are referred [ 0001 ] This application is the Continuation of Interna to herein as a “ BOO3, BOO4 , BOO5 , BOO6 , B007 , BOO8 , tional Patent Application No. PCT /US2018 /040637 , filed BOO9 , BO011 , BOO10 , BO013 , BO014 , BO015 , Jul . 2 , 2018 , which claims the benefit of priority to U.S. BO016 , etc. " , which are also known as ( a.k.a ) “ BOOS3, Provisional Application Nos . 62 / 527,946 , filed Jun . 30 , BOOS4 , BOOS5 , BOOS6 , BOOS7 , BOOS8 , BOOSO , 2017 ; and 62 / 611,858 , filed Dec. 29 , 2017 ; and also a BOOS11 , BOOS10 , BOOS13 , BOOS14 , BOOS15 , Continuation of PCT /US2018 /040646 , filed Jul. 2 , 2018 , BOOS16 , etc." , respectively. Therefore , the media desig which claims the benefit of priority to U.S. Provisional nated as “ BOO ” herein is interchangeably used as “ BOOS ” Application No. 62 / 527,862 , filed Jun . 30 , 2017 ; each of in the present invention . which is herein incorporated by reference in its entirety. [ 0010 ] In some embodiments, the initiation media com TECHNICAL FIELD prise Murashige & Skoog ( MS ) salts , Woody Plant ( WPM ) tissue culture salts , and / or Driver Kuniyuki Walnut ( DKW ) [ 0002 ] This invention provides compositions, systems, tissue culture salts , and sucrose . In some embodiments , the and methods for efficient, rapid and large scale production of concentration of one or more components in the MS , WPM , plants using bioculture in vitro . In some embodiments , the or DKW salts is modified . In some embodiments , the present invention provides compositions, methods, and sys sucrose concentration is about 25 to 35 g / L , for example , tems for the micropropagation and mass production of about 30 g / L . perennials, grasses , monocots, dicots , and phyto -pharma [ 0011 ] In some embodiments , the initiation media com ceutical plants. In some embodiments, the present invention prises at least one cytokinin . In some embodiments, the provides compositions, methods, and systems for the pro initiation media further comprises at least one auxin . duction of virus - free plants. [ 0012 ] In some embodiments, the cytokinin is meta - topo lin (mT ) or functional derivatives thereof. In some embodi BACKGROUND ments , the mT concentration in the initiation media is about [ 0003 ] This invention generally relates to a method of 0.1 to 30 mg / L , for example, about 1-3 mg / L . rapid regeneration / proliferation of plant tissues . More par [ 0013 ] In some embodiments, the auxin is Naphthale ticularly, it relates to an improved method of plant tissue neacetic acid or functional derivatives thereof. In some proliferation which comprises culturing a tissue or an organ embodiments, the NAA concentration in the initiation media of a plant, a part of the same or cultured cells to proliferate is about 0.01 to 1 mg/ L , for example, about 0.1 mg / L . the tissue , organ or cultured cells , thereby regenerating a [ 0014 ] In some embodiments, the auxin is IBA or func plant body or producing a useful substance formed by that tional derivatives thereof. In some embodiments , the IBA plant , for the purposes of rapidly generating plants. concentration in the initiation media is about 0.01 to 1 mg / L , [ 0004 ] The state of the art is such that the demand for for example , about 0.1 mg/ L . plants and plant products far outweight the availability [ 0015 ] In some embodiments , the media further comprises possible with the techniques known in the art . There exists a gibberellin acid . In some embodiments , the gibberellin a clear nead in the art for the rapid proliferation of viable acid is GA3 or functional derivatives thereof. In some plants , and the present application seeks to meet the demand . embodiments, the gibberellin acid concentration in the ini tiation media is about 0.2 to 20 mg / L , for example , about 2 SUMMARY OF THE INVENTION mg / L. [ 0005 ] The present invention provides compositions, [ 0016 ] In some embodiments , the initiation media are methods, kits , bioreactors , and systems for efficient and liquid, semi- liquid , solid , or semi - solid media . In some rapid propagation of pistachio plants at a large scale via embodiments, the initiation media comprise about 4 to about bioculture . 10 grams gelling agent, such as agar. [ 0006 ] In some embodiments, the present invention [ 0017 ] In some embodiments, the initiation media has a describes an automated , or semi- automated , low - cost system pH of about 5.0 to 6.0 , for example, about 5.7 . for the production of pistachio plants, which significantly [ 0018 ] In some embodiments , the multiplication media are increases the quantity and quality of pistachio plants, the similar to , or as the same as the initiation media . number and size of the resulting plants, reduces the cost and [ 0019 ] In some embodiments, the rooting media comprise shortens the cultivation time . Murashige & Skoog ( MS ) salts , Woody Plant ( WPM ) tissue [ 0007 ] This invention provides novel compositions and an culture salts , and / or Driver Kuniyuki Walnut ( DKW ) tissue efficient and rapid system for mass propagation of pistachio culture salts , and sucrose . In some embodiments , the sucrose plants in vitro . concentration is about 25 to 35 g / L , for example , about 30 [ 0008 ] In one embodiment, the present invention provides g / L . In some embodiments, the sucrose concentration is media for plant micropropagation . In some further embodi much higher, for example, at least 60 g / L , such as about 60 ments , the media are used for micropropagation of pistachio g / L to about 120 g / L . In some embodiments, the rooting plants . media comprises at least one auxin . In some embodiments, [ 0009 ] In some embodiments, the media are initiation the rooting media do not comprise any cytokinin . media , multiplication media , and rooting media , such as the [ 0020 ] In some embodiments, the auxin is Indole - 3 -bu BOO3 , BOO4 , BOO5 , BOO6 , B007 , BOO8 , BOO9 , tyric acid ( IBA ) or functional derivatives thereof. In some US 2020/0383331 A1 Dec. 10 , 2020 2 embodiments , the IBA concentration in the rooting media is In some embodiments, the cultures are maintained on the about 0.5 to 5 mg / L , for example, about 1-3 mg /L . In some multiplication medium for 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , or more embodiments, the IBA concentration is much higher, for cycles . In some embodiments , each cycle lasts about a example, about 100 mg / L to about 1500 mg / L , such as about couple of days to a couple of weeks, such as about 3 days 250 mg / L to about 1000 mg / L . to about one week . In some embodiments, the cultures are [ 0021 ] In some embodiments , the rooting media com maintained on the multiplication medium for an additional prises charcoal. In some embodiments , the charcoal has a 30 days , wherein the tissue multiplies by about 2 to about 7 concentration of about 500 mg / L to 1500 mg / L , such as times . about 1000 mg / L . [ 0030 ] In some embodiments, the methods further option [ 0022 ] In some embodiments , the rooting media are liquid , ally comprise ( c ' ) dividing the multiplied plant tissues into semi- liquid , solid , or semi - solid media . In some embodi clumps. In some embodiments , each clump contains about 3 ments , the rooting media comprise at least two types of to 6 shoots . 1 . gelling agents . In some embodiments, at least one gelling [ 0031 ] In some embodiments , the methods further com agent is agar or carrageenan . In some embodiments, the total prise ( d ) transferring the multiplied shoots of step ( c ) or step concentration of gelling agents is about 5 to 10 g / L , for ( c ' ) on a rooting medium to produce a pistachio plant. In example , about 5 g / L . In some embodiments , the media some embodiments , the rooting medium is a BOO8 , B009 , comprising much higher concentration of auxin is a liquid or B0011 , BO010 , BO013 , BO014 , BO015 , or BO016 semi- liquid media. medium . In some embodiments, the rooting medium is a [ 0023 ] In some embodiments, the rooting media has a pH solid medium . In some embodiments , the rooting medium is of about 5.0 to 6.0 , for example, about 5.7 . a liquid medium . In some embodiments, clumps are kept on [ 0024 ] In some embodiments , the initiation medium and / the medium until individual plants with roots are developed . or the multiplication medium comprise MS medium con In some embodiments, the shoot is about 3-4 weeks old . taining double concentration of meso elements ( e.g. , one or [ 0032 ] In some embodiments, the multiplied shoots are more of CaC12.2H20 , MgSO4.7H2O , and KH2PO4 ) , kept on a first rooting medium having a higher sugar double iron , and one or more Gamborg's vitamins ( e.g. , one concentration and then transferred to a second rooting or more of myo - inositol, Nictotinic acid , pyridoxine salts , medium having a lower sugar concentration . In some and thiamine salts ) . embodiments, the first rooting medium has at least about 60 [ 0025 ] The present invention also provides kits for pro g / L sugar first and second rooting medium has about 30 g / L ducing plants . In some embodiments, the kits are used for sugar. In some embodiments, the first rooting medium and producing pistachio plants . In some embodiments , the kits the second rooting medium have about the same sugar comprise one or more initiation medium described herein , concentration . In some embodiments, the shoot is kept on one or more multiplication medium as described herein , the first media having higher sugar concentration for about and / or one or more rooting medium as described herein . 1-3 weeks , and then cultured on the second media having ( 0026 ] The present invention further provides methods for lower sugar concentration for about 2-4 weeks until root producing plants. In some embodiments, the methods are develops. used for producing pistachio plants . [ 0033 ] In some embodiments, the first rooting medium [ 0027 ] In some embodiments, the methods of the present and the second rooting medium has an auxin concentration invention comprise ( a ) obtaining pistachio explant. In some of about 0.5 to 5 mg / L . In some embodiments, the multiplied embodiments , the pistachio explant is selected from the shoots are kept on a first rooting medium having a higher group consisting of single -node explants , shoot tips , basal auxin concentration and then transferred to a second rooting ( bottom ) parts of plants with multiple buds. medium having a lower auxin concentration . In some [ 0028 ] In some embodiments, the methods further com embodiments, the first rooting medium has at least about 30 prise ( b ) initiating shoot from the explant obtained in step ( a ) mg / L auxin and second rooting medium has about 0.5 to 5 on an initiation medium . In some embodiments, the step is mg / L auxin . In some embodiments, the first rooting medium done in a container, e.g. , culture vessels ( such as baby food and the second rooting medium have about the same sugar jars ) with ventilated lids wherein the container contains an concentration . In some embodiments , the shoot is kept on initiation medium . In some embodiments , the initiation the first media having higher auxin concentration for about medium is a BO03 , BOO4 , BOOS , BOO6 , or BOO7 media . 1-24 hours , and then cultured on the second media having The pistachio explant is cultured in the container until the lower auxin concentration for about 2-4 weeks until root explant forms multiple meristematic buds , and the first develops. axillary shoots appear. [ 0034 ] In some embodiments of the present invention , in [ 0029 ] In some embodiments, the methods further com order to verify pathogen - free plants are produced , the meth prise ( c ) multiplying the shoot initiated from step ( b ) on a ods further comprise testing for the presence or absence of multiplication medium . In some embodiments, the step one or more pistachio pathogen species after one or more comprises transferring the materials obtained from step ( b ) cycles . In some embodiments , the pathogen species tested to a new container comprising a multiplication medium of for is a bacteria species , fungal species and / or virus species . the present invention . In some embodiments , the multipli [ 0035 ] In some embodiments, one or more steps described cation medium is a BOO3 , BOO4 , BOO5 , BO06 , or BO07 above are performed in a bioreactor, for example, a tempo medium . In some embodiments, the multiplication medium rary immersion bioreactor. In some embodiments , the tem is a solid medium . In some embodiments , the multiplication porary immersion bioreactor is an ebb and flow bioreactor. medium is a liquid medium . In some embodiments, cultures In some embodiments, the multiplication step and / or rooting are maintained under standard growth condition, such as step is performed in the bioreactor. In some embodiments , about 20 to 28 ° C. ( for example, about 22-24 ° C. ) , under a when a bioreactor is used , one or more of the media day / nigh photoperiod ( for example, a 16/8 day / night cycle ). mentioned above is in liquid or semi- liquid form . The size US 2020/0383331 A1 Dec. 10 , 2020 3 of the bioreactor can be any suitable size based on produc [ 0041 ] The present invention provides compositions, tion requirements . For example , the bioreactor can be about methods, kits , bioreactors , and systems for efficient and 0.1 to about 20 L. The bioreactor can be placed under rapid propagation of yam microtubers at a large scale via standard growth conditions, such as about 20 to 26 ° C. ( for bioculture . example, about 22-24 ° C. ) , and a day /nigh photoperiod ( for [ 0042 ] In some embodiments , the present invention example , a 16/8 day / night cycle ) . In some embodiments, the describes an automated , or semi- automated , low - cost system medium in the bioreactor is refreshed regularly or when for the production of microtubers, which significantly needed . In some embodiments, the medium is refreshed increases the quality of yam plants, the number and size of about every one to about every four weeks (with each round the resulting tubers, and shortens the tuberization stage ( i.e. , called a “ cycle ” ). In some embodiments , after each cycle the time between tuber induction and harvesting ) . amount of biomass increases for about 1 to about 5 times . In [ 0043 ] This invention provides novel compositions and an some embodiments, the pistachio shoots multiplied in the efficient and rapid system for mass propagation of yam bioreactor are transferred to rooting medium to perform step microtubers in vitro , with more than 2 times higher yield of ( d ). In some embodiments, the rooting medium is a liquid microtubers than using presently - available conventional medium . protocols. [ 0044 ] The present invention provides small microtubers [ 0036 ] In some embodiments of the present invention , the ( e.g. , from about 2 mm to about 3 mm in length ) produced methods further comprise propagating the pistachio plants using the micropropagation media , systems and methods of obtained to produce pistachio plants in vitro or in vivo . the present invention , wherein such microtubers produce as [ 0037 ] The present invention also provides methods to much or more total yield of tubers from yam plants produced produce important chemicals derived from pistachio plants . from such microtubers as the total yield of tubers from yam In some embodiments, the methods comprise producing plants produced from larger microtubers ( e.g. , from about 5 pistachio plants by the tissue culture methods of the present mm to about 7 mm in length ) . invention , and then extracting the chemicals from the pis [ 0045 ] In one embodiment, the present invention provides tachio plants. media for plant micropropagation. In some further embodi ments, the media are used for micropropagation of yam [ 0038 ] In some embodiments , the initiation medium and / microtubers . or the multiplication medium comprise MS medium con [ 0046 ] In some embodiments, the media are propagation taining double concentration of meso elements ( one or more and multiplication media . of CaC12.2H20 , MgSO4.7H2O , and KH2PO4 ) , double iron , [ 0047 ] In some embodiments, the media are pre - tuberiza and one or more Gamborg's vitamins ( one or more of tion media . myo - inositol, Nictotinic acid , pyridoxine salts , and thiamine [ 0048 ] In some embodiments, the media are tuberization salts ) . media . [ 0039 ] The present invention also provides methods for [ 0049 ] In some embodiments, the propagation and multi inducing root from a pistachio explant in vitro . In some plication media comprise Murashige & Skoog ( MS ) salts embodiments , the methods comprise growing a pistachio and sucrose . In some embodiments, the sucrose concentra explant on a first rooting medium , and then transferring the tion is about 15 g / L to 25 g / L , for example, about 20 g / L . In pistachio explant to a second rooting medium , wherein the some embodiments, the propagation and multiplication first rooting medium has a higher sugar concentration com medium does not contain any plant hormones or plant pared to the second rooting medium . In some embodiments, growth regulators . the first rooting medium comprises about 60 g / L to about [ 0050 ] In some embodiments, the pre -tuberization media 120 g / L sugar , and wherein the second rooting medium comprise sucrose and comprises about 30 g / L sugar. In some embodiment, the first ( i ) at least one cytokinin and at least one auxin ; rooting medium comprises about the same amount of auxin . ( ii ) at least one growth retardant; or In some embodiments, the pistachio explant is pre - rooted on ( iii ) at least one cytokinin , at least one auxin , and at least one the first rooting medium for about 1 to about 3 weeks before growth retardant. being transferred to the second rooting medium . In some [ 0051 ] In some other embodiments , the sucrose concen embodiments , the pistachio explant is grown on the second tration in the pre - tuberization media is about 25 g / L to 35 rooting medium for about 2 to 4 weeks until roots develop . g /L . [ 0040 ] Alternatively, the methods comprise growing a [ 0052 ] In some embodiments, the at least one cytokinin is pistachio explant on a first rooting medium , and then trans 2ip . In some embodiments, the 2ip concentration in the ferring the pistachio explant to a second rooting medium , pre -tuberization media is about 1 mg / L to 10 mg / L . wherein the first rooting medium has a higher auxin con [ 0053 ] In some embodiments , the at least one auxin is centration compared to the second rooting medium . In some IAA . In some embodiments , the IAA concentration in the embodiments, the first rooting medium comprises about 100 pre -tuberization media is about 0.1 mg/ L to 10 mg/ L . mg / L to 1500 mg/ L IBA , and wherein the second rooting [ 0054 ] In some embodiments , the tuberization media com medium comprises about 0.1 to 10 mg / L IBA . In some prise sucrose and embodiments, the first rooting medium comprises about the ( i ) at least one auxin ; same amount of sugar. In some embodiments , the pistachio ( ii ) at least one growth retardant; or explant is pre - rooted on the first rooting medium for about ( iii ) at least one auxin and at least one growth retardant. 1 to 24 hours before being transferred to the second rooting [ 0055 ] In some embodiments, the sucrose concentration in medium . In some embodiments, the pistachio explant is the tuberization media is about 50 g / L to 100 g / L . grown on the second rooting medium for about 2 to 4 weeks [ 0056 ] In some other embodiments , the tuberization media until roots develop. do not comprise any cytokinin . US 2020/0383331 A1 Dec. 10 , 2020 4
[ 0057 ] In some embodiments, the at least one auxin is In some embodiments, the first medium is a solid , semi NAA . In some embodiments, the NAA concentration in the solid , liquid , or semi- liquid medium comprising MS salts , tuberization media is about 0.01 mg / L to about 0.1 mg/ L . IAA , 2ip , and sucrose . In some further embodiments, the [ 0058 ] In some embodiments of the pre - tuberization concentration of IAA is about 0.1 mg / L to 1 mg / L ; the media and tuberization media of the present invention , the concentration of 2ip is about 1 mg / L to 10 mg / L ; and the growth retardant is a gibberellin acid antagonist. In some concentration of sucrose is about 10 g / L to 40 g / L . further embodiments, the gibberellin acid antagonist is ancymidol. In some additional embodiments , the ancymidol [ 0068 ] In some further embodiments , the first medium is concentration in the pre - tuberization media is about 0.1 a BO018 medium . mg / L to 10 mg/ L . [ 0069 ] In some embodiments of the present invention , the [ 0059 ] In some embodiments of the pre - tuberization second medium comprises MS salts and sucrose without any media and tuberization media of the present invention, the hormones or growth regulators . In some embodiments, the media are solid , semi - solid , liquid , or semi - liquid . sucrose concentration is about 10 g / L to 40 g / L , such as [ 0060 ] In some embodiments of the pre - tuberization about 20 g / L . media and tuberization media of the present invention , the [ 0070 ] In some embodiments, the sterilized sprouts are media have a pH of about 5.5 to 6.2 . grown under about 20 ° C. to 28 ° C. , such as about 24 ° C. , [ 0061 ] The present invention also provides sets ( i.e. , com until pathogen - free yam plants are produced . binations , collections , etc.) of media for producing plants. In some embodiments, the sets of media are used for producing [ 0071 ] In some embodiments of the present invention , the yam microtubers . In some embodiments, each set of media sterilized sprouts are grown under day / night light cycle , with comprises: a day time for about 12 hours to 20 hours , such as about 16 ( 1 ) one or more propagation and multiplication medium as hours . described herein ; [ 0072 ] In some embodiments, the sterilized sprouts are ( 2 ) one or more pre -tuberization medium as described grown under a photon flux density of about 50 umol/ m2 / s to herein ; and 120 umol/ m² / s , such as about 85-100 umol/ m² / s . ( 3 ) one or more tuberization medium as described herein . [ 0073 ] In some embodiments of the present invention , in [0062 ] In some embodiments, the pre -tuberization order to verify pathogen - free plants are produced , the meth medium in the set of media comprises sucrose at concen ods further comprise testing for the presence or absence of tration S1 and the tuberization medium in the set of media one or more yam pathogen species after one or more cycles . comprises sucrose at concentration S2 , wherein si is In some embodiments, the pathogen species tested for is a smaller than S2 . In some embodiments, S1 is about 25 g / L bacteria species , fungal species and / or virus species. to 35 g / L and S2 is about 50 g / L to 100 g / L . [ 0074 ] In some embodiments of the present invention , the [ 0063 ] The present invention also provides kits for pro methods further comprise propagating the pathogen - free ducing plants. In some embodiments, the kits are used for yam sprouts obtained to produce yam plants in vitro or in producing yam microtubers . In some embodiments, the kits vivo . In some embodiments, this involves propagating the comprise one or more pre - tuberization medium described pathogen free yam sprouts in a culture tube or a bioreactor. herein and one or more tuberization medium as described In some further embodiments, the bioreactor is a temporary herein . In some embodiments, the kits further comprise one immersion bioreactor. In some embodiments , the temporary or more propagation and multiplication medium as described herein . In some embodiments , the kits comprise immersion bioreactor is an ebb and flow bioreactor, such as one or more sets of media as described herein . the ones described herein . In some embodiments, the biore [ 0064 ] The present invention further provides methods for actor comprises a solid , semi- solid, liquid , or semi- liquid producing plants . In some embodiments, the methods are medium comprising MS salts and sucrose . used for producing yam microtubers . [ 0075 ] In some embodiments, the medium used in the [ 0065 ] In some embodiments, the methods of the present bioreactors does not have any hormones or growth regula invention comprise obtaining pathogen - free yam sprouts . In tors . In some further embodiments, the sucrose concentra some embodiments , such methods comprise breaking field tion of the media used in the bioreactors is about 10 g / L to tuber dormancy to induce buds . In some further embodi 40 g / L , such as about 20 g / L . In some embodiments, use of ments , the field tuber dormancy is broken naturally, or by the bioreactors produces pathogen - free yam sprouts wherein treatment with GA3 , ethanol, temperature, thiourea , ethyl each plant has about 4-7 axillary buds ( e.g. , embryonic ene chlorohydrins , rindite, carbon disulphide, and / or bro shoots that lie at the junction of the stem and petiole of the moethane . plant ). In some embodiments, each bud would develop into [ 0066 ] In some embodiments of the present invention, the a single plant. In some further embodiments, the multipli induced buds are further grown into sprouts . In some further cation factor of using such bioreactors is about 3x to 10x , embodiments , the sprouts are further sterilized . In some such as about 5x to 6x . In some embodiments, the bioreactor additional embodiments, the sprouts are sterilized in sodium use includes about 4-6 weeks on a solid medium , or about dichloroisocyanurate (NaDCC ), such as 0.5 % solution of 2 weeks to about 3 weeks or, in some other embodiments NaDCC . In some further embodiments , the sterilized sprouts about 2.5 weeks to about 3 weeks , on a liquid medium are cultivated in vitro for one or more cycles until pathogen depending on yam variety. In some embodiments , the yam free sprouts are produced . plants are incubated in a PV1 system . In some embodiments, [ 0067 ] In some embodiments of the present invention , the yam plants are incubated in a PV2 system . sterilized sprouts are cultivated on a first medium for one or [ 0076 ] In some embodiments of the present invention more cycle , and then cultivated on a second medium for one using bioreactors , the sprouts are multiplied under about 20 ° or more cycles until pathogen - free yam plants are produced . C. to 28 ° C. , such as about 24 ° C. US 2020/0383331 A1 Dec. 10 , 2020 5
[ 0077 ] In some further embodiments using bioreactors , the [ 0087 ] The present invention further provides bioreactors sterilized sprouts are grown under day / night light cycle , with for plant propagation . In some embodiments, the bioreactors a day time for about 12 hours to 20 hours , such as about 16 are temporary immersion bioreactors , such as ebb and flow hours . bioreactors . [ 0078 ] In some embodiments, the photon flux density is [ 0088 ] In some embodiments , the temporary immersion about 50 umol /m ° /s to 120 umol/ m² / s, such as about 85-100 bioreactors comprise : umol/ m2 / s when the sprouts are grown in a cultivation tube , [ 0089 ] a growth vessel for incubating plant tissue in a or about 20-100 umol/ m² / s, such as 30-80 umol/ m² / s when sterile or substantially sterile environment; the sprouts are grown in a bioreactor. [ 0090 ] a first media container having a first fluid port and [ 0079 ] In some embodiments of the present invention , the a second fluid port, the first fluid port fluidically coupleable methods further comprise pretreating the yam plants in a to the growth vessel ; bioreactor. In some such embodiments , the bioreactor is a [ 0091 ] a second media container having a first fluid port temporary immersion bioreactor, such as an ebb and flow and a second fluid port, the first fluid port fluidically bioreactor described herein . In some embodiments , the yam coupleable to the growth vessel ; plants are pretreated in a pre -tuberization medium described [ 0092 ] a gas source fluidically coupleable to the second herein , such as the BOO18 , BOO23 , BO019 , BOO20 , fluid port of the first media container and second fluid port BOO24 , or combination thereof. of the second media container; and [ 0093 ] a controller operable in a first operating mode in [ 0080 ] In some embodiments, the duration of such pre which pressurized gas is delivered from the gas source to the treatment is about 1 to 3 weeks . In some embodiments , the first media container to displace a first volume of liquid duration can be shorter or long than 1 to 3 weeks whenever contained therein to the growth vessel , and a second oper it is appropriate or necessary. ating mode in which pressurized gas is delivered from the [ 0081 ] In some embodiments of pretreatment, yam plants gas source to the second media container to displace a are grown under about 20 ° C. to 28 ° C. , such as about 24 ° second volume of liquid contained therein to the growth C. In some embodiments of pretreatment, the sterilized vessel . sprouts are grown under day / night light cycle , with a day [ 0094 ] In some embodiments of the present invention , the time for about 12 hours to 20 hours , such as about 16 hours . controller is operable in a third operating mode in which In some embodiments, yam plants in the pretreatment step liquid contained in the growth vessel is allowed to flow from are grown under a photon flux density of about 20 umol/ m² / s the growth vessel into least one of the first media to 100 umol/ m- / s, such as about 30-80 umol/ m ^ / s . container and the second media container. In some further [ 0082 ] In some embodiments, the methods further com embodiments, the controller is operable in a first incubation prise initiating yam microtubers in a bioreactor. In some sequence in which the third operating mode is executed such embodiments, the bioreactor is a temporary immersion subsequent to the first operating mode . In some additional bioreactor, such as an ebb and flow bioreactor described embodiments, the controller is operable in a second incu herein . In some embodiments of the initiating process , the bation sequence in which the third operating mode is yam plants are grown in a tuberization medium described executed subsequent to the second operating mode . In some herein , such as the BOO21 , BOO25 , BOO26 , BO027 , other embodiments , the controller is further operable in a BOO22 , or combination thereof. In some embodiments of plant propagation mode in which the first incubation the initiating process , the sucrose concentration in the tuber sequence and the second incubation sequence are executed . ization medium is higher than the sucrose concentration in In some additional embodiments, the growth vessel is the pre -tuberization medium described above . In some elevated above the first and second media containers to embodiments of the initiating process , the tuberization allow liquid to flow from the growth vessel into at least one medium has a sugar concentration of about 60 g / L to 80 g / L . of the first media container and the second media container [ 0083 ] In some embodiments, the duration of the initiation in the third operating mode . process is about 3 weeks to 6 weeks or less . In some [ 0095 ] In some embodiments of the present invention, the embodiments of the initiation process , the duration can be bioreactors further comprise a manifold fluidically couple shorter or long than 3 weeks to 6 weeks whenever it is able to the growth vessel , the first media container, and the appropriate or necessary , for example , so long as enough second media container. In some further embodiments , he yam microtubers are produced . manifold is operable to control liquid flow between the growth vessel and the first media container and between the [ 0084 ] In some embodiments of the initiating process , the growth vessel and the second media container. In some yam plants are grown under a temperature lower than the additional embodiments , the growth vessel includes a fluid temperature used in the pretreatment process , and / or a conduit configured to siphon liquid from the growth vessel photon flux density lower than the photon flux density used to at least one of the first media container and the second in the pretreatment process . In some embodiments of the media container. In some other embodiments , the growth initiation process, the plants are grown under a temperature vessel is an ebb and flow bioreactor growth vessel . of about 15 ° C. to 25 ° C. , such as 20 ° C. to 24 ° C. [ 0096 ] In some embodiments of the present invention , the [ 0085 ] In some embodiments of the present invention, the controller is operable to control fluid communication yam plants in the tuberization stage are grown under con between the growth vessel and the first media container, tinuous darkness . between the growth vessel and the second media container, [ 0086 ] In some embodiments of the present invention , the between the gas source and the first media container, and methods for producing yam microtubers comprise utilizing between the gas source and the second media container . one or more sets of medium or one or more kits as described [ 0097 ] The present invention further provides methods for herein . producing plants comprising utilizing the temporary immer US 2020/0383331 A1 Dec. 10 , 2020 6 sion bioreactors as described herein . In some further [ 0105 ] In some embodiments, the solid media is selected embodiments, the temporary immersion bioreactors are used from FIG . 26A or 26B . In some embodiments, the liquid for production of yam microtubers . media is a media selected from FIG . 26A or 26B and without [ 0098 ] The present invention further provides systems for agar. In some embodiments , the solid media and liquid plant propagation . In some embodiments , the systems are for media are the same, except the liquid media does not production of yam microtubers . In some further embodi comprise agar. ments , the systems comprise a temporary immersion biore [ 0106 ] The pulsing media can be selected from FIG . 26B , actor, a yam explant, a pre -tuberization medium , and a such that pulsing media is Pulsing media 1 or Pulsing media tuberization medium . In some additional embodiments , the 2 of FIG . 26B . In some embodiments, the pulsing media is temporary immersion bioreactor is the ebb and flow biore Pulsing media 1 of FIG . 26B , wherein TDZ is substituted actor described herein . In some further embodiments , the with a different cytokinin , such as BAP, Zeatin , CPPU , or yam explant is a pathogen - free yam plants or plant part, such DPU . as a plant seedling . In some other embodiments, the yam [ 0107 ] In some embodiments, the media for rooting is seeding comprises about 4 to 7 axillary buds . selected from FIG . 27. The media for rooting can be B0068 , [ 0099 ] In some embodiments of the present invention , the BO069 , B0070 , or B0071 from FIG . 27 . pre - tuberization medium comprises sucrose and [ 0108 ] Another aspect of the present invention is media ( i ) at least one cytokinin and at least one auxin ; for micropropagating a plant, a perennial, grass , or phyto ( ii ) at least one growth retardant; or pharmaceutical plant and kits comprising the same . In one ( iii ) at least one cytokinin , at least one auxin , and at least one embodiment, the media is selected from FIG . 26A or 26B . growth retardant. In one embodiment, the media is Pulsing media 1 or Pulsing media 2 . [ 0100 ] In some embodiments, the tuberization medium [ 0109 ] Akit of the present invention can comprise a media comprises sucrose and is selected from FIG . 26A or 26B , such as Pulsing media 1 ( i ) at least one auxin ; or Pulsing media 2. In one embodiment, the pulsing media ( ii ) at least one growth retardant; or is Pulsing media 1 , wherein TDZ is substituted with a ( iii ) at least one auxin and at least one growth retardant. different cytokinin , such as BAP, Zeatin , CPPU , or DPU . [0101 ] The present invention also provides methods of The kit can comprise different combinations of media , such producing yam tubers comprising obtaining pathogen - free that the media can be used sequentially. Thus, in some microtubers via in vitro propagation, wherein the microtu embodiments, the kit can further comprise a rooting media , bers are produced by using any one of the methods set forth such as selected from FIG . 27. In some embodiments, the kit herein ; planting the microtubers ; and obtaining yam tubers . comprises a B0054 , BO055 , BO056 , BOO57 , B0058 , In some embodiments of the present invention , such micro BOO59 , BO060 , BOO61 , BOO62 , BO063 , BOO64 , tubers are about 2 mm - about 3 mm long. In other embodi B0065 , BO066 , or BO067 media from FIG . 26A or 26B . ments , the microtubers are about 5 mm - about 7 mm long. [ 0110 ] In some embodiments, the present invention pro [ 0102 ] The present invention also provides methods of vides compositions , methods, and systems for induction of increasing yam tuber production , comprising obtaining somatic embryos from immature lateral buds in bamboo . pathogen - free microtubers via in vitro propagation ; planting [ 0111 ] Accordingly, in one aspect of the present invention , the microtubers; and obtaining yam tubers, wherein the a method for inducing a somatic embryo from an immature microtubers are less than about 5 mm long , and wherein the lateral bud of bamboo comprises: ( a ) incubating the imma weight of yam tuber produced by using the microtubers less ture lateral bud in BOO72 , B0073 , or B0074 media ; ( b ) than about 5 mm long is higher than the weight of yam tuber subculturing material from the lateral bud from ( a ) to fresh produced by using microtubers more than 5 mm long . In media until a pro - embryo structure is induced ; ( c ) transfer some embodiments of the present invention , the microtubers ring the pro - embryo from ( b ) to B0077 or B0078 media are about 2 mm - about 3 mm long . for the pro - embryo to develop into an embryo ; and ( d ) [ 0103 ] The present invention provides compositions, transferring the embryo from ( c ) to B0079 or B0080 methods and systems for plant tissue culture , for example , media for embryo maturation . compositions , methods and systems for plant micropropa [ 0112 ] In one embodiment, the method further comprises gation . In some embodiments, the compositions , methods desicatting or germinating the embryo from ( d ) on B0072 and systems are used for plant in vitro micropropagation . In media . In another embodiment, the B0072 media in step ( d ) some embodiments, the present invention provides compo does not contain a growth regulator. sitions , methods, and systems for the micropropagation and [ 0113 ] In another embodiment, in step ( a ) , the immature mass production of perennials, grasses , and phyto - pharma lateral bud is in B0072 , BO073 , or B0074 media for one ceutical plants. to three days . In another embodiment, in step ( a ) , the [ 0104 ] One aspect of the present invention is a method for immature lateral bud is in B0072 , B0073 , or B0074 micropropagating a plant comprising : ( a ) incubating an media for up to seven days . explant of the plant on a solid media ; ( b ) transferring a [ 0114 ] In some embodiments, in step ( a ) the immature microshoot from the explant of ( a ) to a bioreactor with liquid lateral bud is pulsed with BO075 and BO076 media . In one media ; ( c ) exposing the microshoot to a pulsing media ; ( d ) embodiment, the pulsing with B0075 and B0076 media is harvesting the microshoot at maturity ; and ( e ) transferring for one to three days . In another embodiment, the pulsing the mature microshoot to a media for rooting, wherein the with B0075 and BO076 media is for up to seven days. plant is a perennial, grass, or phyto -pharmaceutical plant. In [ 0115 ] In some embodiments , in step ( b ) , subculturing to some embodiments , the method can further comprise expos fresh media is performed every 28 days. In one embodiment, ing the microshoot to medium comprising meta - topoline the subculturing is performed every 28 days for a period of after step (C ) . about 6 months. US 2020/0383331 A1 Dec. 10 , 2020 7
[ 0116 ] The bamboo can be Phyllostachys edulisi ‘ Moso ' , medium to produce one or more embryos. In some embodi Phyllostachys bissetti, Fargesia denudata, Pleioblastus for ments, the first medium is the medium for initiating bamboo tunei, Sasa Veitchii , Pleioblastus viridistriatus, Thamno somatic embryogenesis as described herein . Non - limiting calamus crassinodus, Chusquea Culeo " Cana Prieta ” , examples of first media are B0072 , B0073 , and B0074 . Bambusa Old Hamii , Phyllostachys Moso , Phyllostachys [ 0128 ] In some embodiments, the explant is obtained from Atrovaginata , Dendrocalamus Asper , Guadua Angustifolia , a juvenile or a mature bamboo plant. In some embodiments, Phylostachys Nigra , Fargesia rufa, Fargesia nitida , Borinda the explant is selected from the group consisting of node Boliana , Fargesia murielae , Pleioblastus fortune, Fargesia segments, immature leaves , immature embryos, and mature robusta , or Bambusa Oldhamii. seeds . In some embodiments, the explant comprises meri [ 0117 ] Another aspect of the present invention is a media stematic cells located in axillary or lateral buds . for inducing a somatic embryo from an immature lateral bud [ 0129 ] In some embodiments, optionally the methods fur of bamboo, wherein the media is B0072 , B0073 , B0074 , ther comprise ( a ' ) culturing the embryo obtained from step B0075 , B0076 , B0077 , B0078 , B0079 , or B0080 . In ( a ) in a pulsing media . Non - limiting examples of pulsing some embodiments, a combination of different media is used media are BO075 and BO076 . In some embodiments , the sequentially . cells are kept on pulsing media for about 1 to 3 days or for [ 0118 ] Also provided herein is a kit comprising a media , up to seven days . In some embodiments, the pulsing media wherein the media is B0072 , B0073 , B0074 , B0075 , is essentially as the same as a first media , except for that the B0076 , B0077 , B0078 , BO079 , or BOO80 . The kit can concentration of one or more components is doubled or comprise different combinations of media , such that the tripled , or more compared to that of a first media . media can be used sequentially. [ 0130 ] In some embodiments, the methods further com [ 0119 ] In some embodiments, provided are media for prise ( b ) culturing the embryo obtained from step ( a ) or ( a ' ) initiating bamboo somatic embryogenesis. In some embodi in second medium to propagate embryonic cells . ments , the media comprise at least one plant growth regu [ 0131 ] In some embodiments , the second medium is a lator . In some embodiments, the media comprise at least one liquid nutrient medium . In some embodiments, the second cytokinin . In some embodiments, the media comprise at medium is a solid nutrient medium . In some embodiments, least one auxin and at least two cytokinins . the solid nutrient media of the present invention comprise [ 0120 ] In some embodiments, the cytokinins are isolated only mT , but not TDZ . In some embodiments, the second or synthetic. In some embodiments, the cytokinins are nutrient media comprise one or more amino acids . In some selected from the group consisting of Nø -benzylaminopurine embodiments, the amino acids are selected from the group ( BAP ) , thidiazuron ( TDZ ) , 2 - isopentenyladenine ( 2 - ip ) , consisting of glutamine , adenine , derivatives thereof, ana kinetin , meta - topolin (mT ) , kinetin , dicamba, 2,4 - D , piclo logs thereof, and any combinations thereof. In some embodi ram , derivatives thereof, analogs thereof, and any combina ments , the amino acids are proline and serine. In some tions thereof. In some embodiments , the concentration of embodiments , the concentration of amino acid is about 0.5 each of the cytokinins ranges from about 0.01 uM to 100 to 2 g / L . Non - limiting examples of second media are UM , or about 0.1 to 10 mg / L , such as about 1 to 5 mg / L . BO077 and BO078 . [ 0121 ] In some embodiments, the media for initiating [ 0132 ] In some embodiments, the second nutrient media bamboo somatic embryogenesis further comprise one or further comprise one or more vitamins . In some embodi more carbon sources . In some embodiments, the carbon ments , the vitamins are selected from the group consisting of sources are selected from the group consisting of sucrose , vitamin A ( e.g. , retinol, pro - vitamin A carotenoids ), vitamin glucose , maltose , lactose , or a combination thereof. B1 ( e.g. , thiamine ), vitamin B2 ( e.g. , riboflavin ), vitamin B3 [ 0122 ] In some embodiments, the media for initiating ( e.g. , niacin ) , vitamin B5 ( e.g. , pantothenic acid ), vitamin bamboo somatic embryogenesis are liquid media , semi B6 ( e.g., pyridoxine ), vitamin B8 ( e.g. , biotin ) , vitamin B9 liquid media , solid , or semi- solid media . ( e.g. , folic acid ) , vitamin B12 ( e.g. , cobalamin ) , vitamin C [ 0123 ] In some embodiments , the media for initiating ( e.g. , ascorbic acid ) , vitamin K ( e.g. , phylloquinone, mena bamboo somatic embryogenesis are solidified by one or quinone ), and any combinations thereof. more gelling agents . In some embodiments , the gelling [ 0133 ] In some embodiments , the second nutrient media agents are selected from the group consisting of agar , comprises casein . carrageenan , gellan gum , alginic acid and its salts , agarose , [ 0134 ] In some embodiments, the second nutrient media and any combinations thereof. In some embodiments , the further comprise one or more carbon source . In some media are solidified by 5 g /L agar. embodiments, the carbon source is selected from the group [ 0124 ] In some embodiments, the media for initiating consisting of sucrose , glucose , maltose , lactose , or a com bamboo somatic embryogenesis further comprise one or bination thereof. more macronutrients, one or more micronutrients, and / or [ 0135 ] In some embodiments, the methods further com one or more vitamins. prise ( c ) transferring and culturing the embryogenic suspen [ 0125 ] In some embodiments, the macronutrients, the sion obtained from step ( b ) onto one or more third media to micronutrients, and the vitamins are those found in the produce mature somatic embryos. In some embodiments, the standard MS media . In some embodiments, amount of one third media are solid media . In some embodiments , the third ore more components in the MS media can be doubled . media comprise MS salts . In some embodiments , amount of [ 0126 ] Also provided are methods for in vitro propagation one ore more components in the MS media can be doubled . of bamboo . In some embodiments, the methods comprise In some embodiments, the third media comprise abscisic propagating bamboo through somatic embryogenesis. In acid ( ABA ). In some embodiments, the concentration of some embodiments, the bamboo is moso bamboo . ABA in the third media is about 1.0 to about 100 uM . [ 012 ] In some embodiments , the methods comprise ( a ) Non - limiting examples of third media are B0079 and culturing an explant obtained from a bamboo plant on a first BOO80 . US 2020/0383331 A1 Dec. 10 , 2020 8
[ 0136 ] In some embodiments, the third media further [ 014 ] In some embodiments , the present invention pro comprise charcoal ( e.g. , active charcoal ). In some embodi vides compositions , methods, and systems for the produc ments, the concentration of the charcoal is about 0.01 % to tion of virus - free plants, such as agricultural plants. The 10% by weight. plant can be a plantlet. [ 0137 ] In some embodiments, the methods further com [ 0147 ] Accordingly, in one aspect of the present invention, prise ( d ) germinating the mature somatic embryos obtained a method for producing a virus - free plant comprises ( a ) from step ( c ) to produce bamboo plants. In some embodi incubating a plant culture with BOO81 medium ; ( b ) sub ments , the mature somatic embryos are germinated on one jecting an explant of the plant culture of ( a ) to thermo or more fourth media . In some embodiments, the third media therapy, wherein the explant grows into a plantlet; ( c ) are solid media . In some embodiments , the fourth media excising an apical meristem from the plantlet of ( b ); ( d ) comprise MS salts , LV salts , or a combination thereof. In placing the apical meristem of ( c ) into a regeneration media ; some embodiments, the fourth media further comprise one wherein a virus - free plantlet is produced from the apical or more amino acids . In some embodiments, the amino acids meristem of ( e) . are selected from the group consisting of glutamine, aspara [ 0148 ] In one embodiment, the culture is incubated under gines , arginine, proline , analogs thereof, and combinations a 16 h light photoperiod at 80-100 umol/ m² / x light intensity thereof. In some embodiments , the concentration of each at 24 ° C. in step ( a ) . The incubation can be for one to two amino acid is about 0.01 to 100 mM . In some embodiments, weeks . the fourth media does not comprise any plant growth regu [ 0149 ] In another embodiment, thermotherapy comprises lator . incubating the explant under a 16 h light photoperiod at [ 0138 ] In some embodiments, one or more or all of the 30-40 umol/ m² / s light intensity at 37 ° C . The thermotherapy media used in the methods described herein are liquid and / or can be for one week . semi- liquid media . In some embodiments, some of the [ 0150 ] In some embodiments , the method comprises media used herein are liquid and / or semi- liquid media , while excising the apical meristem in step ( c ) using a method as the others are solid and / or semi - solid media . depicted as in FIG . 33 . [ 0139 ] In some embodiments, one or more or all steps in [ 0151 ] In some embodiments, step ( d ) comprises incubat the methods described herein are performed in a bioreactor. ing the apical meristem in regeneration media under a 16 h In some embodiments , the bioreactor is a temporary immer light photoperiod at 80-100 umol / m ° / x light intensity at 24 ° sion bioreactor, such as an ebb and flow bioreactor. C. [ 0152 ] In some embodiments , the regeneration media of [ 0140 ] Also provided are kits comprising the initiated step ( d ) is selected from FIG . 32. In other embodiments , the embryogenic tissue or the ire somatic embryos obtained regeneration media of step ( d ) comprises an antiviral, such by the methods described herein . In some embodiments , the as Ribavirain ( also known as Virazole ). kits comprise one or more media of the present invention . [ 0153 ] The method for producing a virus - free plant can [ 0141 ] In some embodiments, the present invention pro also further comprise : ( e ) subculturing the apical meristem vides compositions, methods, and systems for the reduction of step ( d ) , wherein the subculturing can be performed every of a phenolic in plant, such as bamboo . The bamboo may be two to three weeks . in in vitro cultures . [ 0154 ] In some embodiments, the method for producing a [ 0142 ] Accordingly, in one aspect of the present invention , virus - free plant further comprises: ( f) transferring the apical a method for reducing the production of a phenolic in meristem to a regeneration media . In some embodiments, bamboo comprises incubating a bamboo tissue culture , the regeneration media of step ( f) is different than the explant or seed in BOO32 , BOO33 or BOO34 media , regeneration media of step ( d ). In other embodiments, the wherein the bamboo tissue culture, explant, or seed produces regeneration media of step ( f) is the same as the regeneration less of the phenolic as compared to a bamboo tissue culture , media of step ( d ). explant or seed incubated in media that is not BO032 , [ 0155 ] The method for producing a virus - free plant can BO033 or BOO34 . In one embodiment, the phenolic is a also further comprise: ( g ) subculturing the apical meristem , polyphenol. In some embodiments, the phenolic is a luteolin wherein the subculturing can be every two or three weeks . derivative , flavone, flavone glycoside or phenolic acid . [ 0156 ] The plantlet produced by a method disclosed herein [ 0143 ] The bamboo can be Phyllostachys edulisi Moso ', can be subcultured or tested for viruses , such as by ELISA . Phyllostachys bissetti, Fargesia denudata , Pleioblastus for The plantlet can be an agricultural plant, such as potato , tunei, Sasa Veitchii, Pleioblastus viridistriatus, Thamno tomato , yam , sugar beet, cassava , cucumber or cauliflower. calamus crassinodus, Chusquea Culeo " Cana Prieta " , [ 0157 ] Another aspect of the present invention is the Bambusa Old Hamii , Phyllostachys Moso , Phyllostachys media for producing a virus - free plantlet and kits comprising Atrovaginata, Dendrocalamus Asper, Guadua Angustifolia , the same . In one embodiment, the media is selected from Phylostachys Nigra, Fargesia rufa, Fargesia nitida, Borinda FIG . 32. In one embodiment, the media is BOO81 . Boliana, Fargesia murielae , Pleioblastus fortune, Fargesia [ 0158 ] A kit of the present invention can comprise B0081 robusta , or Bambusa Oldhamii. media . The kit can comprise different combinations of [ 0144 ] Another aspect of the present invention is a media media, such that the media can be used sequentially. Thus, for reducing phenolic production by bamboo, wherein the in some embodiments, the kit comprises comprise BOO81 media is BOO32 , BOO33 , or BOO34 . In some embodi media and one or more regeneration media , wherein the ments, a combination of different media is used sequentially. regeneration media can be selected from FIG . 32 . [ 0145 ] Also provided herein is a kit comprising a media , [ 0159 ] This present invention provides compositions , wherein the media is BOO32 , BO033 , or BOO34 . The kit methods, and systems for the production of virus - free potato can comprise different combinations of media , such that the plants. Accordingly, in one aspect of the present invention , media can be used sequentially . a method for producing a virus - free potato plant comprises US 2020/0383331 A1 Dec. 10 , 2020 9
( a ) incubating a potato explant in a plant culture with increases the quantity and quality of hemp plants, the BO081 medium ; ( b ) subjecting an explant of the plant number and size of the resulting plants, reduces the cost and culture of ( a ) to thermotherapy , wherein the explant grows shortens the cultivation time . into a plantlet; ( c ) excising an apical meristem from the [ 0174 ] This invention provides novel compositions and an plantlet of ( b ) ; ( d ) placing the apical meristem of ( c ) into a efficient and rapid system for mass propagation of hemp regeneration media ; wherein a virus - free plantlet is pro plants in vitro . duced from the apical meristem of ( e ) . [ 0175 ] In one embodiment, the present invention provides [ 0160 ] In one embodiment, the culture is incubated under media for plant micropropagation . In some further embodi a 16 h light photoperiod at 80-100 umol /mº / x light intensity ments, the media are used for micropropagation of hemp at 24 ° C. in step ( a ) . The incubation can be for one to two plants. weeks. [ 0176 ] In some embodiments, the media are initiation [ 0161 ] In another embodiment, thermotherapy comprises media, multiplication media , and rooting media , such as the incubating the explant under a 16 h light photoperiod at BOO3 , BOO4 , BOO5 , BO06 , BOO7 , BOO8 , BOO9 , 30-40 umol/ mº / s light intensity at 37 ° C. The thermotherapy BO011 , BOO10 , BOO13 , BO014 , BO015 , BO016 , com can be for one week . bination thereof, or functional equivalents thereof ( e.g. , by [ 0162 ] In some embodiments, the method comprises reducing or increasing one or more component concentra excising the apical meristem in step ( c ) using a method as tion , or by adding or removing one or more component, depicted as in FIG . 33 . wherein the media maintain the same function ). [ 0163 ] In some embodiments , step ( d ) comprises incubat [ 0177 ] In some embodiments, the initiation , multiplica ing the apical meristem in regeneration media under a 16 h tion , and / or rooting media comprise Murashige & Skoog light photoperiod at 80-100 umol/ m- / x light intensity at 24 ° ( MS ) salts , Woody Plant ( WPM ) tissue culture salts , and /or C. Driver Kuniyuki Walnut ( DKW ) tissue culture salts , and [ 0164 ] In some embodiments, the regeneration media of sucrose . In some embodiments, the concentration of one or step ( d ) is selected from FIG . 20. In other embodiments , the more components in the MS , WPM , or DKW salts is regeneration media of step ( d ) comprises an antiviral, such modified . In some embodiments , the sucrose concentration as Ribavirain ( also known as Virazole ). is about 25 to 35 g / L , for example , about 30 g / L . In some [ 0165 ] The method for producing a virus - free plant can embodiments, the sucrose concentration is much higher, for also further comprise : ( e ) subculturing the apical meristem example, at least 60 g / L , such as about 60 g / L to about 120 of step ( d ) , wherein the subculturing can be performed every g / L . two to three weeks . [ 0178 ] In some embodiments, the initiation , multiplica [ 0166 ] In some embodiments, the method for producing a tion , and / or rooting media comprises at least one cytokinin . virus - free plant further comprises: ( f) transferring the apical In some embodiments, the cytokinin is is meta - topolin ( mT ) meristem to a regeneration media . In some embodiments , or any functional derivative . In some embodiments, the mT the regeneration media of step ( f ) is different than the concentration in the initiation media is about 0.5 to 5 mg / L , regeneration media of step ( d ). In other embodiments, the for example , about 1-3 mg/ L . regeneration media of step ( f) is the same as the regeneration [ 0179 ] In some embodiments , the media further comprises media of step ( d ). a gibberellin acid . In some embodiments, the gibberellin acid is GA3 or functional derivatives thereof. In some [ 0167 ] The method for producing a virus - free plant can embodiments, the gibberellin acid concentration in the root also further comprise : ( g ) subculturing the apical meristem , ing media is about 0.2 to 20 mg / L , for example, about 0.5-5 wherein the subculturing can be every two or three weeks . mg / L . [ 0168 ] The plantlet produced by a method disclosed herein [ 0180 ] In some embodiments, the initiation, multiplica can be subcultured or tested for viruses, such as by ELISA . tion , and / or rooting media are liquid , semi- liquid , solid , or [ 0169 ] Another aspect of the present invention is the semi- solid media . In some embodiments, the initiation media for producing a virus - free potato plant and kits media comprise about 4 to about 10 grams gelling agent, comprising the same . In one embodiment, the media is such as agar. selected from FIG . 32. In one embodiment, the media is [ 0181 ] In some embodiments, the initiation multiplication, BOO81 . and /or rooting media has a pH of about 5.0 to 6.0 , for [ 0170 ] Akit of the present invention can comprise B0081 example, about 5.7 . media . The kit can comprise different combinations of [ 0182 ] In some embodiments, the initiation , multiplica media , such that the media can be used sequentially. Thus , tion , and / or rooting medium comprise MS medium contain in some embodiments, the kit comprises comprise BOO81 ing double concentration of meso elements ( e.g. , one or media and one or more regeneration media , wherein the more of CaC12.2H2O , MgSO4.7H2O , and KH2PO4 ) , regeneration media can be selected from FIG . 32 double iron , and one or more Gamborg's vitamins ( e.g. , one [ 0171 ] In some embodiments of the disclosure , the media / or more of myo - inositol, Nictotinic acid , pyridoxine salts , medium is selected from on or more of BOO1 - BOO91 . and thiamine salts ) . [ 0172 ] The present invention provides compositions, [ 0183 ] The present invention also provides kits for pro methods, kits , bioreactors , and systems for efficient and ducing plants. In some embodiments , the kits are used for rapid propagation of hemp plants at a large scale via producing hemp plants or plant part. In some embodiments , bioculture . the kits comprise one or more initiation medium described [ 0173 ] In some embodiments, the present invention herein , one or more multiplication medium as described describes an automated , or semi-automated , low - cost system herein , and / or one or more rooting medium as described for the production of hemp plants, which significantly herein . US 2020/0383331 A1 Dec. 10 , 2020 10
[ 0184 ] The present invention further provides methods for some embodiments, the present invention provides compo producing plants. In some embodiments, the methods are sitions , methods, and systems for the micropropagation and used for producing hemp plants or plant part in vitro . mass production of cannabis plants. [ 0185 ] In some embodiments, the methods of the present [ 0191 ] One aspect of the present invention is a method for invention comprise ( a ) obtaining a hemp explant for hemp micropropagating a plant comprising : ( a ) incubating an culture ; In some embodiments, the hemp explant is selected explant of the plant on a solid media ; ( b ) transferring a from apical and / or lateral buds of shoots of the hemp plants . microshoot from the explant of ( a ) to a bioreactor with liquid ( 0186 ] In some embodiments, apical and lateral buds of media ; ( c ) exposing the microshoot to a pulsing media ; ( d ) plants are washed with a mild detergent and surface steril harvesting the microshoot at maturity; and ( e ) transferring ized under aseptic conditions . Surface sterilization can be the mature microshoot to a media for rooting, wherein the achieved by immersing the shoot buds in solutions of bleach . plant is a cannabis plant and the plant part is a cannabis plant In some embodiments, sterilization process is performed for part. In some embodiments , the method can further comprise a period of time ranging from about a few minutes to about exposing the microshoot to medium comprising meta -topo a couple of hours . In some embodiments, the shoots were line after step ( c ) . In some embodiments of the disclosure , placed in a sterile surface such as the laminar flow hood . In the media /medium is selected from on or more of B001 further embodiments, dead tissues can be removed using a BOO91 . sharp scalpel after surface sterilization . [ 0192 ] In some embodiments, the solid media is selected [ 0187 ] In some embodiments, the methods further com from FIG . 26A or 26B . In some embodiments , the solid prise ( b ) initiating the hemp culture with the explant from media is BOO54 , BO055 , BO056 , BOO57 , BO058 , step ( a ) on an initiation medium . In some embodiments, the BO059 , BO060 , BOO61 , B0062 , BO063 , BO064 , step is done in a test tubes and / or a container such as culture BOO65 , BO066 or B0067 media . In some embodiments, vessels ( such as baby food jars ) with ventilated lids wherein the liquid media is a media selected from FIG . 26A or 26B the container contains an initiation medium . In some and without agar . In some embodiments, the solid media and embodiments, the initiation medium is a BOO3 , B004 , liquid media are the same , except the liquid media does not BOO5 , BO06 , or BOO7 media . In some embodiments, the comprise agar . initiation medium is a solid medium . In some embodiments, [ 0193 ] The pulsing media can be selected from FIG . 26B , the initiation medium is a liquid medium . In some embodi such that pulsing media is Pulsing media 1 or Pulsing media ments , the initiation step ( b ) further comprises inoculating 2 of FIG . 26B . In some embodiments, the pulsing media is the explant on the initiation medium in vitro . Pulsing media 1 of FIG . 26B , wherein TDZ is substituted [ 0188 ] In some embodiments, the methods further com with a different cytokinin , such as BAP, in , CPPU , or prise ( c ) multiplying the initiated hemp culture from step ( b ) DPU . on a multiplication medium . In some embodiments, the step comprises transferring the materials obtained from step ( b ) [ 0194 ] In some embodiments , the media for rooting is to a new container comprising a multiplication medium of selected from FIG . 27. The media for rooting can be B0068 , the present invention . In some embodiments, the multipli BOO69 , BO070 , or BOO71 from FIG . 27 . cation medium is a BOO3 , BOO4 , BOO5 , BO06 , or BOO7 [ 0195 ] Another aspect of the present invention is media medium . In some embodiments , the initiation medicume is for micropropagating a cannabis plant and kits comprising the same as multiplication medium . In some embodiments, the same . the multiplication medium is a solid medium . In some [ 0196 ] In one embodiment, the media is selected from embodiments, the multiplication medium is a liquid FIG . 26A or 26B . In one embodiment, the media is Pulsing medium . In some embodiments, cultures are maintained media 1 or Pulsing media 2 of FIG . 26B . In one embodi under standard growth condition , such as about 20 to 28 ° C. ment, the pulsing media is Pulsing media 1 of FIG . 26B , ( for example, about 23-27 ° C. ) , under a day /nigh photope wherein TDZ is substituted with a different cytokinin , such riod ( for example, a 16/8 day /night cycle ) . In some embodi as BAP, Zeatin , CPPU , or DPU . The media can comprise ments, the cultures are maintained on the multiplication different combinations of media , such that the media can be medium for 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , or more cycles . In some used sequentially. Thus , in some embodiments, the media embodiments, each cycle lasts about a couple of days to a can further comprise a rooting media, such as selected from couple of weeks , such as about four weeks to about eight FIG . 27. In some embodiments, the media comprises a weeks . In some embodiments, about 5 plants up to about BO054 , BOO55 , BO056 , BOO57 , BOO58 , BO059 , 15,000 plants per the culture vessel are produced for each B0060 , BO061 , B0062 , BO063 , B0064 , BO065 , cycle . In some embodiments , size and volume of a culture BO066 , or BO067 media from FIG . 26A or 26B . vessel for multiplying initiated hemp culture step range from [ 0197 ] A kit of the present invention can comprise a media about 5 ml to about 50 gallons , for example, about 100 ml is selected from FIG . 26A or 26B , such as Pulsing media 1 to about 10 gallans . or Pulsing media 2 of FIG . 26B . In one embodiment, the [ 0189 ] In some embodiments, the methods further com pulsing media is Pulsing media 1 of FIG . 26B , wherein TDZ prise ( d ) transferring the multiplied hemp culture of step ( c ) is substituted with a different cytokinin , such as BAP , Zeatin , on a rooting medium to produce a hemp plant with root . In CPPU , or DPU . The kit can comprise different combinations some embodiments, the rooting medium is a BOO3 , BOO4 , of media, such that the media can be used sequentially . Thus , BOOS , BOO6 , or BOO7 medium . in some embodiments, the kit can further comprise a rooting [ 0190 ] The present invention provides compositions , media , such as selected from FIG . 27. In some embodi methods and systems for plant tissue culture, for example , ments , the kit comprises a BOO54 , BO055 , BO056 , compositions , methods and systems for plant micropropa BOO57 , BO058 , BOO59 , B0060 , BO061 , BOO62 , gation . In some embodiments, the compositions , methods BO063 , B0064 , BO065 , BO066 , or BO067 media from and systems are used for plant in vitro micropropagation . In FIG . 26A or 26B . US 2020/0383331 A1 Dec. 10 , 2020 11
[ 0198 ] In some embodiments of the disclosure , the media / [ 0224 ] FIG . 24 is a side view of the portion of the medium is selected from on or more of BOO1 - BOO91 . oscillating rack of FIG . 14 , in a second configuration . [ 0225 ] FIG . 25 is a side view of the portion of the BRIEF DESCRIPTION OF THE DRAWINGS oscillating rack of FIG . 14 , in a third configuration . [ 0199 ) FIG . 1 depicts shoot tip necrosis in pistachio plant [ 0226 ] FIG . 26A and FIG . 26B are tables showing media cultured in vitro on BOO12 medium . formulations of BOO54 , BO055 , BO056 , BOO57 , BO058 , [ 0200 ] FIG . 2 depicts that new shoots developed from BO059 , BO066 , and BOO61 ( FIG . 26A ) , and BO062 , axillary buds of single - node explants of pistachio after 3 BOO63 , BOO64 , B0065 , BO066 , and BO067 ; and puls days of culture on BOO3 medium . ing media ( FIG . 26B ) ; which may be used for micropropa [ 0201 ] FIG . 3 depicts root development in pistachio (Pis gation of aloe vera , ginger, grape, cannabis, garlic, onion , tacia atlantia x Pistacia intergerrina ) plants cultures on Echinacea, miscanthus , arundo donax , switch grass , rice, rooting medium BO010 for 2 weeks. The image on the right sugar cane , echinacea , geranium , and other plants disclosed is a close - up of the left image . The arrows point to the new herein . root . [ 0227 ] FIG . 27 is a table showing rooting media for [ 0202 ] FIG . 4 depicts well- developed, healthy pistachio micropropagation of plants. plants growing in vitro on BOO3 medium . [ 0228 ] FIG . 28 is a photograph of Echinacea plants ready [ 0203 ] FIG . 5 is a block diagram of an example of one to be planted . system of the invention . [ 0229 ] FIG . 29 is a photograph of established [ 0204 ] FIG . 6 is a schematic illustration of a non - limiting Hakonechloa plant in the greenhouse. embodiment of the system of FIG . 5 . [ 0230 ] FIG . 30 is a table showing medium recipes for [ 0205 ] FIG . 7 is a schematic illustration of a media con induction , establishment and maturation of Moso somatic tainer of the system of FIG . 6 . embryos. DT- 200 = Thidiazuron ; St - 10 =meta - topolin [ 0206 ] FIG . 8 is a schematic illustration of a manifold of [ 0231 ] FIG . 31 is a table showing medium recipes for the system of FIG . 6 . BOO32 , BO033 , and BOO34 , which are media that reduce [ 0207 ] FIG . 9A is a front view of a growth vessel of the phenolic production by bamboo tissue cultures. system of FIG . 6 . [ 0232 ] FIG . 32 is a table showing medium recipes for [ 0208 ] FIG.9B is a side view of the growth vessel of FIG . producing virus - free plants. BOO81 medium contains an 9A . antiviral ribavirin ( Virazole ), while the remaining media, [ 0209 ] FIG . 9C is a top view of the growth vessel of FIG . referred to as “ regeneration media ” can be used for culturing 9A . meristems, such as apical meristems from plantlets that were [ 0210 ] FIG . 10 is a flowchart of a plant propagation treated with antiviral chemicals and / or thermotherapy. sequence of the invention . BO082 and B0083 can be used for potato , while the other [ 0211 ] FIG . 11 is a table depicting illustrative composi media are named according to the plant in which the media tions and media for in vitro propagation and tuberization of can be used for. yams and other plants . [ 0233 ] FIG . 33 is schematic of meristem excision from [ 0212 ] FIG . 12 is a table depicting illustrative composi young potato shoots in vitro . tions of additional media for in vitro tuberization of yams. [ 0234 ] FIG . 34A , FIG . 34B , FIG . 34C , and FIG . 34D are [ 0213 ] FIG . 13 is a table showing medium recipes of photographs of meristemic potato clones from Russet Bur BO031 , BOO48 , BO049 , DKW , BOO51 , BOO50 , BOO52 , bank FIG . 34A - 34B , Mazama ( FIG . 34C ) , and Yukon Gold and B0053 . All components are represented in milligrams ( FIG . 34D ) potatoes . per / L except sugar which is represented in grams per / L . [ 0235 ] FIG . 35 is a photograph of plant regeneration from [ 0214 ] FIG . 14 is a front perspective view of an oscillating Mazama meristemic clones . rack , according to an embodiment. [ 0236 ] FIG . 36 is a photograph of plant regeneration from [ 0215 ] FIG . 15 is a side perspective view of the oscillating Yukon Gold meristemic clones . rack of FIG . 14 . [ 0237 ] FIG . 37 is a photograph of plant regeneration from [ 0216 ] FIG . 16 is an enlarged exploded view of a portion Russet Burbank 2 meristemic clones . of the oscillating rack labeled as Region Z in FIG . 15 . [ 0238 ] FIG . 38 is a table showing composition of media [ 0217 ] FIG . 17 is a cross - sectional view of an upright for propagation and rooting of plant culture in vitro , such as included in the oscillating rack of FIG . 8 , taken along line pistachio , hemp and other plants disclosed herein . 4-4 in FIG . 14 . [ 0218 ] FIG . 18 is a perspective view of a shelf assembly DETAILED DESCRIPTION included in the oscillating rack of FIG . 14 . [ 0239 ] Pistachio is a member of the Anacardiaceae family [ 0219 ] FIG . 19 is a perspective view of a portion of the in the Pistacia genus . It is referred to as the “ green gold tree " shelf assembly of FIG . 18 . due to its high economic value . [ 0220 ] FIG . 20 is a cross - sectional view of a platform [ 0240 ] The plant material used in this study was interspe included in the portion of the shelf assembly taken along line cific hybrid UCB I ( P. atlantica x P. intergerrina ) from 7-7 in FIG . 19 . California , which is the result of a closed pollination of the [ 0221 ] FIG . 21 is a perspective view of bushings included single P. atlantica tree with P. integerrima pollen . This P. in the shelf assembly of FIG . 17 . atlantica x P. intergerrina hybrid has increased vigor, as [ 0222 ] FIG . 22 is an exploded view of a drive assembly compared with P. atlantica , and generally equal or greater included in the oscillating rack of FIG . 14 . vigor than P. intergerrina . In addition , the UCB I is more [ 0223 ] FIG . 23 is a side view of a portion of the oscillating resistant to Verticillium vilt , which is a common soil - borne rack of FIG . 14 , in a first configuration . fungal disease of pistachio rootstocks in California . US 2020/0383331 A1 Dec. 10 , 2020 12
[ 0241 ] The information on micropropagation of pistachio [ 0249 ] Though meristem tissue culture has been success plants in vitro is very limited , and refers only to P. vera L. fully applied in potato for development of virus free plants Moreover , those few published protocols for growing pis and virus free potato tuber plants, there is continuing need tachio plants in vitro failed to provide satisfactory growth of for improvement in methods of producing virus - free potato our cultivar. Thus, it is virtually new and unexplored area . In plants. The present invention meets this need and provides addition , rooting of pistachio presents a major obstacle for related advantages. the pistachio industry, as well as in vitro shoot tip necrosis . [ 0250 ] The main problems associated with microtuber [ 0242 ] Here , we describe a highly efficient system for production are low yield of tubers ( usually less than 1 tuber propagation of pistachio plants in vitro , which provided easy per plant ), small tuber size ( usually less than 1 cm in scale - up of high quality, healthy plants within short period diameter ), long production time ( usually 4-6 months ), and of time . higher labor costs . Thus, there is a continuing need for improvement of microtuber production, which the present [ 0243 ] Yams are generally grown from seed yams — these invention addresses. are tubers specifically grown to be disease free and provide [ 0251 ] With increasing burdens on land to produce food consistent and healthy plants . To be disease free , the areas and biomass for energy and materials additional attention is where seed yams are grown are selected with care . In the being placed on identifying and utilizing faster growing and USA this restricts production of seed yams to only 15 states more productive plants . Although many plants are suitable out of the 50 states that grow yams. These locations are for such purposes , there is still a great need to develop selected for their cold hard winters that kill pests and long compositions, methods, and systems for fast, economical sunshine hours in the summer for optimum growth . plant propagation . [ 0244 ] Virus and viroid diseases are among the most significant diseases in yam seed production and certification . They include Dioscorea bacilliform virus ( DB V , genus Definition Badnavirus ), Yam mosaic virus ( YMV, genus Potyvirus ), [ 0252 ] As used herein , the verb “ comprise” as is used in and Yam mild mosaic virus ( YMMV, genus Potyvirus ). Viral this description and in the claims and its conjugations are disease is an important reason attributed to lower yield of used in its non - limiting sense to mean that items following yam varieties. Other diseases , such as bacterial and fungal the word are included , but items not specifically mentioned diseases also present a problem for yam production . are not excluded . [ 0245 ] Meristem tissue culture has been successfully [ 0253 ] The term “ a ” or “ an ” refers to one or more of that applied in yams for development of virus free plants and entity ; for example, " a gene” refers to one or more genes or at least one gene . As such , the terms “ a ” ( or “ an ” ), “ one or virus free yam tuber plants. more ” and “ at least one” are used interchangeably herein . In [ 0246 ] Yam microtubers offer several advantages over addition , reference to “ an element” by the indefinite article seed yams and meristem tissue culture for the seed produc “ a ” or “ an” does not exclude the possibility that more than tion of yams, including relatively shorter field times neces one of the elements are present, unless the context clearly sary to supply commercial growers ( 3 or 4 years compared requires that there is one and only one of the elements . with 7 or more years ), and greatly improved seed tuber [ 0254 ] As used herein , the term “ plant” refers to any living quality ( fewer viral, bacterial, fungal problems) ( see Donelly organism belonging to the kingdom Plantae ( i.e. , any genus / et al . , 2003 ) . In contrast to in vitro propagated plants, species in the Plant Kingdom ) . to trees, herbs, bushes , microtubers can be stored and transplanted directly to the grasses , vines , ferns , mosses and green algae . The term field without an acclimatization stage . Because of relatively refers to both monocotyledonous plants, also called mono small size , their handling, storage and transport are more cots , and dicotyledonous plants , also called dicots . practical than that of regular tubers or in vitro plants. Many Examples of particular plants include but are not limited to countries lacking isolated and pathogen - free growing areas bamboo , corn , potatoes , roses, apple trees , sunflowers, that permit the production of yam seed tubers use microtuber wheat, rice , bananas, tomatoes , opo , pumpkins , squash , propagation as a vital component for yam production . lettuce, cabbage , oak trees , guzmania, geraniums, hibiscus , [ 0247 ] Potatoes are generally grown from seed potatoes clematis , poinsettias, sugarcane , taro, duck weed , pine trees, these are tubers specifically grown to be disease free and Kentucky blue grass , zoysia , coconut trees, brassica leafy provide consistent and healthy plants . To be disease free , the vegetables ( e.g. broccoli, broccoli raab , Brussels sprouts, areas where seed potatoes are grown are selected with care . cabbage, Chinese cabbage ( Bok Choy and Napa ) , cauli In the USA this restricts production of seed potato to only flower, cavalo , collards, kale , kohlrabi, mustard greens, rape 15 states out of the 50 states that grow potatoes. These greens, and other brassica leafy vegetable crops ), bulb locations are selected for their cold hard winters that kill vegetables ( e.g. garlic , leek , onion ( dry bulb , green , and pests and long sunshine hours in the summer for optimum Welch ), shallot , and other bulb vegetable crops ) , citrus fruits growth . ( e.g. grapefruit, lemon , lime , orange , tangerine, citrus [ 0248 ] Virus and viroid diseases are among the most hybrids, pummelo , and other citrus fruit crops ), cucurbit significant diseases in potato seed production and certifica vegetables ( e.g. cucumber, citron melon , edible gourds , tion . They include Potato leaf roll virus ( PLRV ), Potato virus gherkin , muskmelons ( including hybrids and / or cultivars of A (PVA ), Potato virus M ( PVM ) , Potato virus S ( PVS ) , cucumis melons ) , water -melon , cantaloupe, and other cucur Potato virus X ( PVX ), Potato virus Potato virus S ( PVS ) , bit vegetable crops ) , fruiting vegetables ( including eggplant, Potato virus X (PVX ), Potato virus Y (PVY ) and Potato ground cherry , pepino, pepper, tomato , tomatillo , and other spindle tuber viroid ( PSTVd ). Viral disease is an important fruiting vegetable crops ), grape, leafy vegetables ( e.g. reason attributed to lower yield of potato varieties . Other romaine ), root/ tuber and corm vegetables ( e.g. potato ) , and diseases , such as bacterial and fungal diseases also present tree nuts ( almond , pecan , pistachio , and walnut ), berries a problem for potato production . ( e.g. , tomatoes , barberries, currants, elderberries, gooseber US 2020/0383331 A1 Dec. 10 , 2020 13 ries , honeysuckles, mayapples, nannyberries, Oregon [ 0259 ] In some embodiments, the compositions , methods , grapes, see -buckthorns , hackberries, bearberries, lingonber and systems are useful for propagation of phyto - pharma ries , strawberries, sea grapes, lackberries , cloudberries , ceutical plants . A phyto -pharmaceutical plant is a plant that loganberries, raspberries, salmonberries , thimbleberries , and can be used for a plant- based medicament. In one embodi wineberries ), cereal crops ( e.g. , corn , rice , wheat, barley, ment, one or more active ingredients in a phyto -pharmaceu sorghum , millets, oats, ryes , triticales, buckwheats, fonio , tical is derived from a plant disclosed herein . In some and quinoa ), pome fruit ( e.g. , apples, pears ), stone fruits embodiments, the active ingredient is a plant disclosed ( e.g. , coffees, jujubes, mangos, olives , coconuts, oil palms , herein . pistachios , almonds , apricots, cherries , damsons, nectarines , [ 0260 ] In some embodiments, the compositions, methods, peaches and plums ), vine ( e.g. , table grapes , wine grapes ), and systems are useful for propagation of Aloe vera , Ginger, fiber crops ( e.g. hemp, cotton ) , ornamentals, and the like. Grape, Cannabis, Garlic, Onion, Echinacea , Geranium , For example, the plant is a species in the tribe of Camelin Hakonechloa, Miscanthus, Arundo donax , Switch grass , eae , such as C. alyssum , C. anomala , C. grandiflora , C. Rice , or Sugar cane . hispida, C. laxa , C. microcarpa , C. microphylla , C. per [ 0261 ] In some embodiments, the compositions, methods, sistens, C. rumelica , C. sativa , C. Stiefelhagenii, or any and systems are useful for bamboo plant in vitro propaga hybrid thereof. For example, in some embodiments, the tion . As used herein , the term “ bamboo ” refers to plants in plant is a species in the Pistachioa genus . In some embodi the subfamily of Bambusoideae . Representative genera of ments, the plant is W. japonica . In some embodiments , the bamboo are described in International Patent Application plant is a species in the Solanum genus , such as S. tuberosum Publication No. WO2011100762 , which is incorporated S. stenotomum , S. phureja, S. goniocalyx , S. ajanhuiri. S. herein by reference in its entirety. chaucha , S. juzepczukii , and S. curtilobum .In some embodi [ 0262 ] As used herein , the terms “ herb ” , “ herbs ” and ments, the plant is a yam variety of the S. tuberosum species . " herbal” all refer to an annnnual , biennial, or perennial plant [ 0255 ] In some embodiments , the compositions , methods , that does not develop persistent woody tissue but dies down and systems are useful for crop plant in vitro propagation . In at the end of a growing season . Herbal plants typically are some embodiments, a crop plant is an agricultural plant. As capable of flowering and producing seeds . In some contexts used herein , the term “ crop plant ” refers to any plant grown the terms refer to a plant or plant part valued for its for any commercial purpose , including, but not limited to the medicinal, savory , or aromatic qualities. Examples of herbs following purposes : seed production , hay production , orna include , but are not limited to , sage , rosemary, parsley, basil , mental use , fruit production , berry production , vegetable catnip and marijuana . production , oil production , protein production , forage pro [ 0263 ] As used herein , " herbal medicine ” or “ herbal duction , animal grazing , golf courses , lawns , flower produc medicinal” refer to herbs, herbal materials , herbal prepara tion , landscaping, erosion control, green manure , improving tions , and finished herbal products that contain parts of soil tilth / health, producing pharmaceutical products / drugs , plants, other plant materials , or combinations thereof as producing food or food additives, smoking products, pulp active ingredients . Herbs include crude plant material, for production and wood production. For example , an agricul example , leaves , flowers , fruit, seed , and stems. Herbal tural plant can be potato , tomato , yam , sugar beet , cassava , materials include , in addition to herbs, fresh juices , gums, cucumber, or cauliflower. fixed oils , essential oils , resins , and dry powders of herbs . [ 0256 ] In some embodiments, the compositions, methods, Herbal preparations are the basis for finished herbal products and systems are useful for monocotyledon plants propaga and may include comminuted or powdered herbal materials, tion . As used herein , the term “ monocotyledon ” or “ mono or extracts, tinctures , and fatty oils of herbal materials . cot” refer to any of a subclass (Monocotyledoneae ) of Finished herbal products consist of herbal preparations made flowering plants having an embryo containing only one seed from one or more herbs. See , e.g. , Perspectives in Clinical leaf and usually having parallel - veined leaves, flower parts Research , April - June 2016 , 7 ( 2 ) : 59-61 . in multiples of three , and no secondary growth in stems and [ 0264 ] As used herein , the term “ phytopharmaceutical ” roots . Examples include lilies ; orchids; rice ; corn , grasses , (aka “ phyto -pharmaceutical ") refers to a pharmaceutical of such as tall fescue , goat grass , and Kentucky bluegrass ; plant origin . grains, such as wheat, oats and barley ; irises; onions and [ 0265 ] As used herein , the term “ plant part ” refers to any palms . part of a plant including but not limited to the shoot , root , [ 0257 ] In some embodiments , the compositions, methods, stem , axillary buds , seeds , stipules , leaves , petals, flowers, and systems are useful for propagation of perennials. The ovules , bracts , branches, petioles , node , internodes, bark , perennial can be an evergreen , deciduous, monocarpic, pubescence , tillers , rhizomes, fronds, blades , pollen , stamen , woody, or herbaceous perennial. In some embodiments, the microtubers , and the like . The two main parts of plants perennial is Begonia, banana , goldenrod , mint, agave , maple grown in some sort of media , such as soil , are often referred tree , pine tree , apple tree , alfalfa or red clover. to as the “ above - ground ” part, also often referred to as the [ 0258 ] In some embodiments, the compositions, methods, “ shoots ” , and the “ below - ground ” part, also often referred to and systems are useful for propagation of grasses . The grass as the “ roots ” . can be of the Poaceae ( or Gramineae ), Cyperaceae or [ 0266 ] As used herein , the term “ germplasm ” refers to the Juncaceae family. The grass can be a perennial grass or a genetic material with its specific molecular and chemical cereal grass . The grass can be switchgrass, big bluestem , makeup that comprises the physical foundation of the miscanthus, alfalfa, orchard grass , or reed canarygrass . The hereditary qualities of an organism . grass can be bamboo , tall fescue, goat grass , or Kentucky [ 0267 ] As used herein , the phrase " derived from ” refers to bluegrass. Other types of grasses include wheat, rye , oat , the origin or source , and may include naturally occurring, barley, soy, and hemp, as well as straws derived therefrom . recombinant, unpurified , or purified molecules . A nucleic US 2020/0383331 A1 Dec. 10 , 2020 14 acid or an amino acid derived from an origin or source may [ 0275 ] As used herein , the term “ genotype ” refers to the have all kinds of nucleotide changes or protein modification genetic makeup of an individual cell , cell culture, tissue , as defined elsewhere herein . organism ( e.g. , a plant ), or group of organisms. [ 0268 ] As used herein , the term “ offspring ” refers to any [ 0276 ] As used herein , the term “ clone” refers to a cell , plant resulting as progeny from a vegetative or sexual group of cells , a part, tissue , organism ( e.g. , a plant ), or reproduction from one or more parent plants or descendants group of organisms that is descended or derived from and thereof. For instance an offspring plant may be obtained by genetically identical or substantially identical to a single cloning or selfing of a parent plant or by crossing two parent precursor. In some embodiments, the clone is produced in a plants and include selfings as well as the F1 or F2 or still process comprising at least one asexual step . further generations. An F1 is a first - generation offspring [ 0277 ] As used herein , the term “ hybrid ” refers to any produced from parents at least one of which is used for the individual cell , tissue or plant resulting from a cross between first time as donor of a trait, while offspring of second parents that differ in one or more genes. generation ( F2 ) or subsequent generations ( F3 , F4 , etc.) are [ 0278 ] As used herein , the term “ inbred ” or “ inbred line” specimens produced from selfings of Fi's , F2's etc. An F1 refers to a relatively true - breeding strain . may thus be ( and usually is ) a hybrid resulting from a cross [ 0279 ] As used herein , the term “ population ” means a between two true breeding parents ( true -breeding is genetically homogeneous or heterogeneous collection of homozygous for a trait ), while an F2 may be ( and usually is ) plants sharing a common genetic derivation . an offspring resulting from self - pollination of said F1 [ 0280 ] As used herein , the term “ bioreactor” refers to any hybrids. vessel , device or system capable of holding , supporting [ 0269 ] As used herein , the term “ plant tissue ” refers to any and / or growing viable tissue . In other words, the term part of a plant. Examples of plant organs include , but are not “ bioreactor ” as used herein may refer to a growth vessel that limited to the leaf, stem , root , tuber, seed , branch , pubes holds viable plant tissue , various other components internal cence , nodule, leaf axil , flower, pollen , stamen , pistil , petal , or external to the growth vessel that are required for or aid peduncle, stalk , stigma , style , bract , fruit, trunk , carpel, the holding , supporting and / or growing of viable plant sepal, anther, ovule , pedicel , needle , cone , rhizome, stolon , tissue , and any subsystem thereof. shoot, pericarp , endosperm , placenta , berry, stamen , and leaf [ 0281 ] As used herein , the phrase “ temporary immersion sheath . bioreactor” refers to any bioreactor designed to temporarily [ 0270 ] As used herein , the term “ variety ” or “ cultivar ” wet a part or entire culture or plant tissue with nutrient means a group of similar plants that by structural features medium ( e.g. , liquid or semi - liquid ) followed by draining a and performance can be identified from other varieties part or all of the excess nutrient medium . within the same species . The term “ variety ” as used herein [ 0282 ] As used herein , a " plant propagation system ” is a has identical meaning to the corresponding definition in the bioreactor for growing viable plant tissue . International Convention for the Protection of New Varieties [ 0283 ] As used herein , the media named “ BOO ” is equiva of Plants ( UPOV treaty ), of Dec. 2 , 1961 , as Revised at lent to “ BOOS . ” The media designated as “ BOO ” herein is Geneva on Nov. 10 , 1972 , on Oct. 23 , 1978 , and on Mar. 19 , interchangeably used as “ BOOS ” in the present invention . 1991. Thus, “ variety ” means a plant grouping within a single For example, the media of the present invention are referred botanical taxon of the lowest known rank , which grouping, to herein as a “ BOO3, BOO4 , BOO5 , BOO6 , BOO7 , BOO8 , irrespective of whether the conditions for the grant of a BOO9 , BO011 , BO010 , BO013 , BOO14 , BO015 , breeder's right are fully met , can be i ) defined by the BO016 , etc. " , which are also known as ( a.k.a ) “ BOOS3 , expression of the characteristics resulting from a given BOOS4 , BOOS5 , BOOS6 , BOOS7 , BOOS8 , BOOSI , genotype or combination of genotypes, ii ) distinguished BOOS11 , BOOS10 , BOOS13 , BOOS14 , BOOS15 , from any other plant grouping by the expression of at least BOOS16 , etc. " , respectively . one of the said characteristics and iii ) considered as a unit [ 0284 ] More rapidly growing plants are available for cul with regard to its suitability for being propagated tivation and industrial uses , including plants in the subfam unchanged . ily Bambusoidea . The subfamily Bambusoideae ( of the [ 0271 ] As used herein , the term “ cross ” , “ crossing ” , “ cross family Poaceae ) , comprises both woody and herbaceous pollination ” or “ cross - breeding ” refer to the process by bamboos. At present roughly 120 genera of temperate and which the pollen of one flower on one plant is applied tropical woody bamboos are recognized . Bamboos are ver ( artificially or naturally ) to the ovule ( stigma ) of a flower on satile plants with many different applications. It has been another plant. estimated that approximately 2.2 billion people worldwide use bamboo to some extent, and in 1985 the global revenue [ 0272 ] As used herein , the term “ cultivar ” refers to a attributable to bamboo was estimated around U.S. $ 4.5 variety , strain or race of plant that has been produced by billion . The market for bamboo is also expanding. Bamboo horticultural or agronomic techniques and is not normally shoots are a staple of Asian cuisine , and bamboo is found in found in wild populations. a number of products including toothpicks, brooms, poles [ 0273 ] As used herein , the term “ cross ” , “ crossing ”, “ cross for viticulture and arboriculture , landscaping materials , par pollination ” or “ cross - breeding ” refer to the process by quet flooring, laminate materials, furniture , handicrafts and which the pollen of one flower on one plant is applied other household items. In addition , bamboo is becoming an ( artificially or naturally ) to the ovule ( stigma ) of a flower on important source of textile material as a component of paper another plant. production and as a source of structural timber . [ 0274 ] As used herein , the term " cultivar” refers to a [ 0285 ] Plant species such as bamboo are considered envi variety, strain or race of plant that has been produced by ronmentally friendly “ green ” products , which have horticultural or agronomic techniques and is not normally extremely rapid growth rates . Despite rapid growth rates, found in wild populations. other characteristics of these plants make it difficult to US 2020/0383331 A1 Dec. 10 , 2020 15 rapidly propagate these plants at an industrial scale . For tages of in vitro culture in a liquid media are often coun example , many commercially important bamboos only terbalanced by technical problems such as asphyxia, hyper flower at intervals of as long as 60-130 years . Compounding hydricity , shear forces and the need for complex equipment. the difficulties of this long flowering cycle is the fact that [ 0292 ] International Patent Application Publication No. many bamboos exhibit mass ( or gregarious) flowering, with WO / 2011 / 100762 , which is incorporated herein by refer all plants in the population flowering simultaneously . For ence in its entirety, describes compositions and methods that example , Phyllostachys bambusoides flowers at an interval are useful for bamboo in vitro propagation . of 130 years , and in this species all plants of the same stock [ 0293 ] The present application discloses novel composi flower at the same time , regardless of differences in geo tions , methods , and systems for the rapid in vitro propaga graphic locations or climatic conditions. After flowering, the tion of plants. The present application also discloses novel bamboo dies . Bamboo's lengthy flowering interval and compositions, methods, and systems for the reduction of propensity for mass flowering makes it very difficult to phenolic production by plants, such as bamboo . obtain seeds for propagation. Compounding this problem is [ 0294 ] The present invention provides compositions and the fact that bamboo seeds , even when they are available, methods that can significantly increase plant tissue culture remain viable for no more than 3-6 months . multiplication rate within a shorter time . In some embodi [ 0286 ] As a result of these difficulties with the propagation ments , a strong cytokinin such as thidiazuron is utilized for of bamboo and other fast growing plant species using a very brief period of time in either a solid or liquid traditional sexual reproduction, these plants are often propa induction medium to induce multiple shoot bud formation in gated by asexual techniques such as clump division and explants of plant species . This bud induction treatment cutting. These asexual propagation techniques , however , are utilizing a media containing a strong cytokinin such as insufficient to meet projected world demand because both thidiazuron is followed by a shoot elongation and mainte their capacity to produce mass scale production, and their nance treatment whereby a relatively weaker cytokinin such practical efficiency, are too low . In addition many asexual as BAP, meta - topolin , 2ip , zeatin and or zeatin riboside is propagation methods have the downside of failing to elimi used to accomplish the shoot elongation and maintenance of nate pathogens present in the parent plants. Therefore , the culture . This process , when alternated methodically compositions , methods, and systems to achieve large scale resulted in culture multiplication rates between 2x and 28x production of plants are highly desirable . Micropropagation within a 3 - week culture cycle . ( also known as tissue culturing with the terms used inter changeably herein ), is an excellent potential method that Pistachio could be used to achieve this aim . [ 0295 ] The pistachio , Pistacia vera in the Anacardiaceae [ 0287 ] Micropropagation is not unlike growing plants family, is a small tree originally from Greater Iran which from cuttings. However, unlike plants grown from cuttings , now can also be found in regions of Syria , Lebanon , Turkey , micropropagated plants are grown in vitro in sterile media . Greece , Tunisia, Kyrgyzstan , Tajikistan , Turkmenistan , Typically, the growth media comprises a solid or semi - solid India , Pakistan , Egypt, Italy ( Sicily ) , Uzbekistan , Afghani material such as agar, with the addition of various com stan , and the United States . The tree produces an important pounds such as nutrients, inorganic salts, growth regulators , culinary nut . sugars , vitamins and other compounds. [ 0296 ] Pistacia vera often is confused with other species [ 0288 ] A benefit of tissue culturing plants is that the plants in the genus Pistacia that are also known as pistachio . These can be grown in a sterile environment so that they may more species can be distinguished from P. vera by their geo likely remain disease free . Other benefits include the ability hic distributions ( in the wild ) and their nuts which are to grow very large numbers of plants in a small space , the much smaller, have a strong flavor of turpentine, and have a reduced water and nutrient needs of micropropagated plants , shell that is not hard . and the rapid multiplication of tissues that can in turn be [ 0297 ] As used herein , the term pistachio refers to all used to yield more tissue culture material . Moreover micro species in the genus Pistacia . In some embodiments, the propagation is very flexible and rapid upscaling is possible pistachio is the hybrid species produced from cross of (within 1 year nearly one million plants can be produced Pistacia atlantica and Pistacia intergerrina . In some from a single genotype ). Such short time frames and large embodiments, the pistachio plant is the Kerman variety. numbers cannot be rivaled by any conventional method . [ 0298 ] Pistachio is a desert plant, and is highly tolerant of Tissue culturing also provides for the production of high saline soil . It has been reported to grow well when irrigated quality plants which are easy to transport and deliver . with water having 3,000-4,000 ppm of soluble salts . Pista [ 0289 ] Some papers have published which address tissue chio trees are fairly hardy in the right conditions , and can culturing of plants . In practice , however ( i.e. , for large or survive temperatures ranging between -10 ° C. ( 14 ° F. ) in mass scale propagation of bamboos ), the compositions, winter and 48 ° C. ( 118 ° F. ) in summer . They need a sunny methods, and systems described in these papers do not position and well - drained soil . Pistachio trees do poorly in translate into commercially viable propagation systems . conditions of high humidity , and are susceptible to root rot [ 0290 ] The difficulties encountered in tissue culturing of in winter if they get too much water and the soil is not plant species include high incidences of endogenous or sufficiently free - draining. Long, hot summers are required surface contaminations and browning, factors related to for proper ripening of the fruit. dormancy or topophysis and hyperhydricity . The present [ 0299 ] Iran , the United States and Turkey are the major disclosure provides compositions , methods, and systems producers of pistachios. The trees are planted in orchards , that overcome these difficulties allowing the commercial and take approximately seven to ten years to reach signifi scale asexual production of plants . cant production. Production is alternate bearing or biennial [ 0291 ] Micropropagation in liquid culture media increases bearing, meaning the harvest is heavier in alternate years . nutrient uptake and promotes growth . However, the advan Peak production is reached at approximately 20 years . Trees US 2020/0383331 A1 Dec. 10 , 2020 16 are usually pruned to size to make the harvest easier . ous flattish or globose seeds . Exemplary yam varieties for Harvesting in the United States and in Greece is often which the present invention applies include , but are not accomplished by using shaking equipment to shake the nuts limited to , white yam , yellow yam , Kokoro yam , water yam , off the tree . After hulling and drying, pistachios are sorted winged yam , purple yam , Chinese yam , air potato , lesser according to open mouth and closed mouth shell . Sun drying yam , bitter yam , and cush - cush yam . has been found to be the best method of drying. Then they [ 0305 ] Depending on the species , yam grows for six to ten are roasted or processed by special machines to produce months and is dormant for two to four months, these two pistachio kernels. phases usually corresponding to the wet season and the dry [ 0300 ) Pistachio trees are vulnerable to a wide variety of season . For maximum yield the yam requires an annual diseases , including but not limited to , Alternaria late blight, rainfall of over 1,500 mm distributed uniformly throughout Armillaria root rot , Aspergillus fruit rot , Blossom and shoot the growing season . White , yellow and water yams normally blight, Camarosporium shoot and panicle blight, Cotton root produce annually a single large tuber, often weighing 5-10 rot , Eutypa dieback , Gum canker, Leaf spot , Panicle and kg . shoot blight, Phomopsis shoot blight, Powdery mildew , [ 0306 ] Yams yield abundantly with little effort, and adapt Phytophthora root and crown rot , Rust, Sclerotinia shoot readily to diverse climates as long as the climate is cool and blight, Seedling blight, Septoria leaf spot, Stigmatomycosis , moist enough for the plants to gather sufficient water from Thread blight, Phytophthora trunk and bark canker, Verti the soil to form the starchy tubers . However, yams do not cillium wilt . Among these is infection by the fungus Botry keep very well in storage and are vulnerable to molds that osphaeria, which causes panicle and shoot blight ( i.e. , kills feed on the stored tubers , quickly turning them rotten . The flowers and young shoots ) , and can damage entire pistachio major problems presently facing yam production are its high orchards. labor requirement, its low yield compared to crops such as [ 0301 ] In California , almost all female pistachio trees are cassava or sweet potato , the relatively large amount of the cultivar “ Kerman " . A scion from a mature female planting material that is required and its long growing Kerman is grafted onto a one - year - old rootstock . Male season . pistachios may be a different variety. [ 0302 ] Pistachios in particular significantly reduced levels Potato of low - density lipoprotein ( LDL cholesterol) while increas [ 0307 ] There are about five thousand potato varieties ing antioxidant levels in the serum of volunteers. Other worldwide . Three thousand of them are found in the Andes health benefits of pistachio include , but are not limited to , alone , mainly in Peru , Bolivia , Ecuador, Chile , and Colom reducing LDL ( “bad ” ) cholesterol and increase the good bia . They belong to eight or nine species, depending on the HDL cholesterol after only a short period of regular con taxonomic school . Apart from the five thousand cultivated sumption; high in antioxidants such as vitamins A and E ; varieties , there are about 200 wild species and subspecies , fighting inflammation , protecting blood vessels and reducing many of which can be cross -bred with cultivated varieties, risk of heart disease . preventing Type 2 diabetes , rich source which has been done repeatedly to transfer resistances to of vitamin B6 , having wide -ranging effects on the nervous certain pests and diseases from the gene pool of wild species system , having lutein and zeaxanthin ( function as protective to the gene pool of cultivated potato species . antioxidants , defending tissues from damage from free radi [ 0308 ] The major species grown worldwide is Solanum cals ) ; helping the body make healthy red blood cells , and tuberosum ( a tetraploid with 48 chromosomes ), and modern helping maintain the health of lymphoid glands, such as the varieties of this species are the most widely cultivated . There thymus, spleen and lymph nodes, ensuring the production of are also four diploid species ( with 24 chromosomes ): S. white blood cells that defend the body from infections. stenotomum , S. phureja, S. goniocalyx, and S. ajanhuiri. There are two triploid species ( with 36 chromosomes ): S. Yams chaucha and S. juzepczukii. There is one pentaploid culti [ 0303 ] Yam is the common name for some plant species in vated species (with 60 chromosomes ): S. curtilobum . There the genus Dioscorea ( family dioscoreaceae ) which produce are two major subspecies of Solanum tuberosum : andigena, tubers, bulbils , or rhizomes having medicinal and economic or Andean ; and tuberosum , or Chilean . The Andean potato is importance . These are perennial herbaceous vines cultivated adapted to the short - day conditions prevalent in the moun for the consumption of their starchy tubers in Africa, Asia , tainous equatorial and tropical regions where it originated . Latin America , the Caribbean , and Oceania . The Chilean potato, native to the Chiloé Archipelago , is [ 0304 ] True yams are botanically distinct from the sweet adapted to the long -day conditions prevalent in the higher potato , but moist - fleshed varieties of sweet potato are often latitude region of southern Chile . called yams in the United States . D. bulbifera, the air -potato [ 0309 ] Most modern potatoes grown in North America yam , is one of the few true yams cultivated for food in the arrived through European settlement and not independently United States. Yams have thick tubers ( generally a devel from the South American sources . However, at least one opment of the base of the stem ) , from which protrude long , wild potato species , Solanum fendleri, is found as far north slender, annual, climbing stems bearing leaves , which are as Texas and used in breeding for resistance to a nematode either alternate or opposite and either entire or lobed and species that attacks cultivated potatoes. A secondary center unisexual flowers in long clusters . The flowers are generally of genetic variability of the potato is Mexico , where impor small and individually inconspicuous, though collectively tant wild species that have been used extensively in modern showy. Each consists of a greenish , bell -shaped or flat breeding are found , such as the hexaploid Solanum demis perianth of six pieces , enclosing six or fewer stamens in the sum , as a source of resistance to the devastating late blight male flowers and surmounting a three - celled , three -winged disease . Another relative native to this region, Solanum ovary in the female flowers . The ovary ripens into a mem bulbocastanum , has been used to genetically engineer the branous capsule , bursting by three valves to liberate numer potato to resist potato blight. US 2020/0383331 A1 Dec. 10 , 2020 17
[ 0310 ] Potatoes yield abundantly with little effort, and America , the Cornell Potato Varieties List , the Canadian adapt readily to diverse climates as long as the climate is Registry of Potato Varieties, the UPOV potato varieties cool and moist enough for the plants to gather sufficient collection , etc. water from the soil to form the starchy tubers . Potatoes do [ 0315 ] Exemplary potato varieties for which the present not keep very well in storage and are vulnerable to molds invention applies include , but are not limited to , Adirondack that feed on the stored tubers, quickly turning them rotten . Blue , Adirondack Red , Agata , Almond , Apline , Alturas, By contrast, grain can be stored for several years without Amandine , Annabelle , Anya , Arran Victory, Atlantic, Ava much risk of rotting. lanche , Bamberg, Bannock Russet, Belle de Fontenay, [ 0311 ] Potato contains vitamins and minerals , as well as BF - 15 , Bildtstar, Bintje, Blazer, Busset , Blue Congo , Bon an assortment of phytochemicals, such as carotenoids and notte , British Queens, Cabritas, Camota , Canela Russet, natural phenols. Chlorogenic acid constitutes up to 90 % of Cara , Carola , Chelina , Chiloé , Cielo , Clavela Blanca , the potato tuber natural phenols. Others found in potatoes Desiree, Estima , Fianna , Fingerling, Flava , German Butter are 4 - O - caffeoylquinic acid ( crypto - chlorogenic acid ) , 5-0 ball , Golden Wonder, Goldrush , Home Guard , Innovator, caffeoylquinic ( neo - chlorogenic acid ), 3,4 - dicaffeoylquinic Irish Cobbler, Jersey Royal , Kennebec , Kerr's Pink , Kestrel, and 3,5 - dicaffeoylquinic acids . [ 58 ] A medium - size 150 g Keuka Gold , King Edward , Kipfler, Lady Balfour, Langlade , ( 5.3 oz ) potato with the skin provides 27 mg of vitamin C Linda, Marcy, Marfona, Maris Piper , Marquis , Megachip , ( 45 % of the Daily Value ( DV ) , 620 mg of potassium ( 18 % Monalisa , Nicola , Pachacoña , Pike , Pink Eye , Pink , Fir of DV ) , 0.2 mg vitamin B6 ( 10 % of DV ) and trace amounts Apple, Primura, Ranger Russet, Ratte, Record , Red LaSoda , of thiamin , riboflavin , folate , niacin , magnesium , phospho Red Norland, Red Pontiac, Rooster, Russet Burbank , Russet rus , iron, and zinc . The fiber content of a potato with skin ( 2 Norkotah , Selma , Shepody, Sieglinde, Silverton, Russet, g ) is equivalent to that of many whole grain breads , pastas , Sirco , Snowden , Spunta , Stobrawa, Superior, Vivaldi, and cereals . Vitelotte , Yellow Finn , Yukon Gold , blue potato varieties [ 0312 ] In terms of nutrition , the potato is best known for ( e.g. , Cream of the Crop ) , Igorota , Solibao , Ganza , Eliane, its carbohydrate content ( approximately 26 grams in a BelRus, Centennial Russet, CenturyRusset, Frontier Russet, medium potato ) . The predominant form of this carbohydrate Hilite Russet, Krantz , Lemhi Russet, Nõoksack , Norgold is starch . A small but significant portion of this starch is Russet, Norking Russet, Ranger Russet, Russet Burbank , resistant to digestion by enzymes in the stomach and small Russet Norkotah , Russet Nugget, Allegany, Atlantic , Beacon intestine, and so reaches the large intestine essentially intact . Chipper, CalWhite, Cascade , Castile , Chipeta , Gemchip , This resistant starch is considered to have similar physi Irish Cobbler, Itasca , Ivory Crisp , Kanona , Katandin , Ken ological effects and health benefits as fiber: It provides bulk , nebec , Kennebec Story , La Chipper, Lamoka, Monona , offers protection against colon cancer , improves glucose Monticello , Norchip , Norwis, Onaway , Chieftain , LaRouge , tolerance and insulin sensitivity, lowers plasma cholesterol NorDonna, Norland , Red La Soda , Red Pontiac , Red Ruby, and triglyceride concentrations, increases satiety , and pos Sangre , Viking, Ontario , Pike, Sebago , Shepody, Snowden , sibly even reduces fat storage . The amount of resistant starch Superior, Waneta, White Pearl , White Roseand, Mazama , in potatoes depends much on preparation methods . Cooking and all genetically modified varieties. More potato varieties and then cooling potatoes significantly increases resistant are described in Clough et al . , Hort Technology, 2010 , starch . For example, cooked potato starch contains about 7 % 20 ( 1 ) : 250-256 ; Potato Variety Handbook, National Institute resistant starch , which increases to about 13 % upon cooling . of Agricultural Botany, 2000 ; Chase et al . , North American [ 0313 ] Potato has been bred into many standard or well Potato Variety Inventory, Potato Association of America , known varieties, each of which has particular agricultural or 1988 , each of which is incorporated by reference in its culinary attributes. In general, varieties are categorized into entirety . a few main groups, such as russets, reds , whites , yellows [ 0316 ] Traditional potato growth has been divided into ( also called Yukons) and purples based on common char five phases . During the first phase , sprouts emerge from the acteristics. For culinary purposes, varieties are often seed potatoes and root growth begins . During the second , described in terms of their waxiness . Floury, or mealy photosynthesis begins as the plant develops leaves and ( baking ) potatoes have more starch ( 20-22 % ) than waxy branches . In the third phase stolons develop from lower leaf ( boiling ) potatoes ( 16-18 % ). The distinction may also arise axils on the stem and grow downwards into the ground and from variation in the comparative ratio of amylose and on these stolons new tubers develop as swellings of the amylopectin . In some embodiments , the potato variety of the stolon This phase is often ( but not always ) associated with present invention is a White Rounds potato variety, a Red flowering . Tuber formation halts when soil temperatures Rounds potato variety, or a Russet potato variety. reach 80 ° F. ( 26.7 ° C. ) ; hence potatoes are considered a [ 0314 ] In some embodiments, the potato is a variety cool - season crop . Tuber bulking occurs during the fourth deposited in the International Potato Center based in Lima , phase , when the plant begins investing the majority of its Peru , which holds an ISO - accredited collection of potato resources in its newly formed tubers. At this stage , several germplasm . The international Potato Genome Sequencing factors are critical to yield : optimal soil moisture and Consortium announced in 2009 that they had achieved a temperature , soil nutrient availability and balance , and resis . draft sequence of the potato genome. The potato genome tance to pest attacks. The final phase is maturation : The plant contains 12 chromosomes and 860 million base pairs mak canopy dies back , the tuber skins harden , and their sugars ing it a medium - sized plant genome . More than 99 percent convert to starches . of all current varieties of potatoes currently grown are direct [ 0317 ] Potato can be used to produce alcoholic beverages , descendants of a subspecies that once grew in the lowlands food for human and domestic animals. The potato starch can of south - central Chile . In some other embodiments, the be used in the food industry as thickeners and binders of potato is a variety included in the European Cultivated soupsand sauces , inthe textileindustry as adhesives , and for Potato Databased ( ECPD ) , the Potato Association of the manufacturing of papers and boards . Waste potatoes can US 2020/0383331 A1 Dec. 10 , 2020 18 be used to produce polylactic acid for plastic products , or [ 0324 ] Cannabis plants produce a unique family of ter used as a base for biodegradable packaging . Potato skins, peno -phenolic compounds called cannabinoids . Cannabi along with honey , are a folk remedy for burns. noids , terpenoids, and other compounds are secreted by glandular trichomes that occur most abundantly on the floral Cannabis calyxes and bracts of female plants. As a drug it usually [ 0318 ] Cannabis, more commonly known as marijuana, is comes in the form of dried flower buds ( marijuana ), resin a genus of flowering plants that includes at least three ( hashish ), or various extracts collectively known as hashish species , Cannabis sativa , Cannabis indica , and Cannabis oil . There are at least 483 identifiable chemical constituents ruderalis as determined by plant phenotypes and secondary known to exist in the cannabis plant (Rudolf Brenneisen , metabolite profiles . In practice however, cannabis nomen 2007 , Chemistry and Analysis of Phytocannabinoids ( can clature is often used incorrectly or interchangeably. Canna nabinoids produced by cannabis) and other Cannabis Con bis literature can be found referring to all cannabis varieties stituents , In Marijuana and the Cannabinoids, ElSohly, ed .; as " sativas” or all cannabinoid producing plants as " indi incorporated herein by reference ) and at least 85 different cas ” . Indeed the promiscuous crosses of indoor cannabis cannabinoids have been isolated from the plant ( El - Alfy, breeding programs have made it difficult to distinguish Abir T , et al . , 2010 , “ Antidepressant -like effect of delta - 9 varieties, with most cannabis being sold in the United States tetrahydrocannabinol and other cannabinoids isolated from having features of both sativa and indica species . Cannabis sativa L ” , Pharmacology Biochemistry and [ 0319 ] Cannabis is one of the world's oldest and most Behavior 95 ( 4 ) : 434-42 ; incorporated herein by reference ). useful cultivated genus of plants. Humans have used hemp http://en.wikipedia.org/wiki/Cannabiscite_note-26 The varieties of cannabis for the production of industrial mate two cannabinoids usually produced in greatest abundance rials , including food , paper , textiles , plastics , detergents, and are cannabidiol ( CBD ) and / or Aº -tetrahydrocannabinol biofuels. Humans also have a long history of using psycho ( THC ) . THC is psychoactive while CBD is not . See , active varieties of cannabis for medical and recreational ElSohly, ed . (Marijuana and the Cannabinoids, Humana applications. Cannabis has long been used for drug and Press Inc. , 321 papers , 2007 ) , which is incorporated herein industrial purposes , fiber (hemp ), for seed and seed oils , for by reference in its entirety , for a detailed description and medicinal purposes , and as a recreational drug . Industrial literature review on the cannabinoids found in marijuana . hemp products are made from Cannabis plants selected to [ 0325 ] Cannabinoids are the most studied group of sec produce an abundance of fiber . Some Cannabis strains have ondary metabolites in cannabis. Most exist in two forms, as been bred to produce minimal levels of THC , the principal acids and in neutral ( decarboxylated ) forms. The acid form psychoactive constituent responsible for the psychoactivity is designated by an “ A ” at the end of its acronym ( i.e. associated with marijuana. Marijuana has historically con THCA ) . The phytocannabinoids are synthesized in the plant sisted of the dried flowers of Cannabis plants selectively as acid forms, and while some decarboxylation does occur bred to produce high levels of THC and other psychoactive in the plant, it increases significantly post -harvest and the cannabinoids . Various extracts including hashish and hash kinetics increase at high temperatures. ( Sanchez and Ver oil are also produced from the plant. poorte 2008 ) . The biologically active forms for human [ 0320 ] Interest in psychoactive varieties of cannabis has consumption are the neutral forms. Decarboxylation is usu recently exploded following the relaxation drug laws within ally achieved by thorough drying of the plant material the United States, and with the discovery of previously followed by heating it , often by either combustion , vapor unrecognized applications for cannabis in the treatment of ization , or heating or baking in an oven . Unless otherwise human diseases such as diabetes, epilepsy , schizophrenia , noted , references to cannabinoids in a plant include both the and cancer. acidic and decarboxylated versions ( e.g. , CBD and CBDA ) . [ 0321 ] Cannabis is diploid , having a chromosome [ 0326 ] The cannabinoids in cannabis plants include , but complement of 2n = 20 , although polyploid individuals have are not limited to , 19 - Tetrahydrocannabinol ( 49 - THC ) , been artificially produced . The first genome sequence of A8 - Tetrahydrocannabinol (18 - THC ), Cannabichromene Cannabis, which is estimated to be 820 Mb in size , was ( CBC ) , Cannabicyclol ( CBL ) , Cannabidiol ( CBD ) , Canna published in 2011 by a team of Canadian scientists ( Bakel et bielsoin ( CBE ) , Cannabigerol ( CBG ) , Cannabinidiol al , “ The draft genome and transcriptome of Cannabis ( CBND ) , Cannabinol ( CBN ) , Cannabitriol ( CBT ) , and their sativa ” Genome Biology 12 : R102 ) . propyl homologs , including, but are not limited to canna [ 0322 ] All known strains of Cannabis are wind - pollinated bidivarin ( CBDV ) , 19 - Tetrahydrocannabivarin ( THCV ) , and the fruit is an achene . Most strains of Cannabis are short cannabichromevarin ( CBCV ) , and cannabigerovarin day plants , with the possible exception of C. sativa subsp . ( CBGV ). See Holley et al . ( Constituents of Cannabis sativa sativa var . spontanea ( = C . ruderalis ), which is commonly L. XI Cannabidiol and cannabichromene in samples of described as " auto - flowering ” and may be day -neutral . known geographical origin , J. Pharm . Sci. 64 : 892-894 , [ 0323 ] The genus Cannabis was formerly placed in the 1975 ) and De Zeeuw et al . ( Cannabinoids with a propyl side Nettle ( Urticaceae ) or Mulberry ( Moraceae ) family, and chain in Cannabis, Occurrence and chromatographic behav later, along with the Humulus genus ( hops ), in a separate ior, Science 175 : 778-779 ) , each of which is herein incorpo family , the Hemp family ( Cannabaceae sensu stricto ). http : // rated by reference in its entirety for all purposes . Non - THC en.wikipedia.org/wiki/Cannabiscite_note-schultes2001a cannabinoids can be collectively referred to as “ CBs ” , 21 Recent phylogenetic studies based on cpDNA restriction wherein CBs can be one of THCV, CBDV , CBGV, CBCV , site analysis and gene sequencing strongly suggest that the CBD , CBC , CBE , CBG , CBN , CBND , and CBT cannabi Cannabaceae sensu stricto arose from within the former noids . Celtidaceae family, and that the two families should be [ 0327 ] In addition to cannabinoids, cannabis also produces merged to form a single monophyletic family, the Canna over 120 different terpenes ( Russo 2011 , Taming THC : baceae sensu lato . potential cannabis synergy and phytocannabinoid -terpenoid US 2020/0383331 A1 Dec. 10 , 2020 19 entourage effects , British Journal of Pharmacology , 163: [ 0331 ] Although , some cannabis varieties will flower 1344-1364 ) . Within the context and verbiage of this docu without the need for external stimuli, most varieties have an ment the terms ' terpenoid ' and ‘ terpene' are used inter absolute requirement for inductive photoperiods in the form changeably. Examples of representative terpines include, but of short days or long nights to induce fertile flowering. The are not limited to , terpinolene, alpha phelladrene, beta first sign of flowering in cannabis is the appearance of ocimene, carene, limonene, gamma terpinene, alpha pinene, undifferentiated flower primordial along the main stem of alpha terpinene, beta pinene, fenchol, camphene, alpha the nodes. At this stage , the sex of the plants are still not terpineol, alpha humulene , beta caryophyllene, linalool, cary distinguishable . As the flower primordia continue to oxide, and myrcene. develop, female (pistillate ), and male ( staminate ) flowers [ 0328 ] Cannabinoids are odorless, so terpenoids are can be distinguished. responsible for the unique odor of cannabis , and each variety [ 0332 ] For most cannabinoid producing purposes , only has a slightly different profile that can potentially be used as female plants are desired . The presence of male flowers is a tool for identification of different varieties or geographical considered undesirable as pollination is known to reduce the origins of samples ( Hillig 2004. “ A chemotaxonomic analy cannabinoid yield , and potentially ruin a crop . For this sis of terpenoid variation in Cannabis ” Biochem System and reason , most cannabis is grown “ sinsemilla ” through veg Ecology 875-891 ) . It also provides a unique and complex etative ( i.e. , asexual ) propagation . In this way, only female flavor smell , and effect profile for each variety that is plants are produced and no space is wasted on male plants. appreciated by both novice users and connoisseurs . In addi [ 0333 ] Commercial production of these medicinal and tion to many circulatory and muscular effects , some terpenes recreational cannabis varieties however, has been slowed interact with neurological receptors . A few terpenes pro down by the lack of true - breeding psychoactive genetics . duced by cannabis plants also bind weakly to Cannabinoid Indeed , most popular cannabis strains in the market do not receptors. Some terpenes can alter the permeability of cell have fixed genetics, and are unable to produce uniform membranes and allow in either more or less THC , while progeny when propagated through seeds . Modern cannabis other terpenes can affect serotonin and dopamine chemistry production techniques thus rely on asexual cuttings of single as neurotransmitters . Terpenoids are lipophilic , and can cannabis “ mother ” plants to produce uniform crops of interact with lipid membranes, ion channels , a variety of genetically identical plants . Current asexual reproduction different receptors including both G - protein coupled odor techniques however, still represent a major bottleneck in ant and neurotransmitter receptors ), and enzymes . Some are cannabis production yields . Improper techniques and incor capable of absorption through human skin and passing the rect hormones and nutrient formulations result in low propa blood brain barrier. gation yields , and slow rooting and recovery of successful [ 0329 ] Cannabis is an annual, dioecious , flowering herb . clones. Although asexual reproduction of cannabis is some The leaves are palmately compound or digitate , with serrate what easily performed , its inherent constraints of time , space leaflets . Cannabis normally has imperfect flowers, with and resources severely limits the total number of plants that staminate “ male ” and pistillate “ female ” flowers occurring can be produced in large scale commercial operations . on separate plants . It is not unusual, however, for individual Furthermore , asexual reproduction of cannabis is constantly plants to separately bear both male and female flowers ( i.e. , plagued by a host of other problems, including , but not have monoecious plants ). Although monoecious plants are limited to , abiotic disorders ( e.g. , nutrition , light quality and often referred to as " hermaphrodites , " true hermaphrodites quantity, water availability, etc. ); pathogens ( e.g. , Powdery ( which are less common in cannabis) bear staminate and Mildew and Pythium root rots ); mites ( e.g. , two spotted pistillate structures on individual flowers, whereas monoe spider mites and hemp russet mite ) ; aphids ( e.g. , rice root cious plants bear male and female flowers at different aphid and hop aphid ); white flies ; viruses ( e.g. , Tobacco locations on the same plant. Mosaic Virus ) and fungus gnats . [ 0330 ] The life cycle of cannabis varies with each variety [ 0334 ] The present disclosure generally relates to compo but can be generally summarized into germination ( or root sitions , systems , and methods for cannabis tissue culture and ing/ recovery after asexual propagation ), vegetative growth , the Cannabis cells , calli , tissues , plant parts and whole and reproductive stages . Because of heavy breeding and plants produced and /or regenerated from such tissue culture . selection by humans, most cannabis seeds have lost dor The disclosures of the present invention circumvent many of mancy mechanisms and do not require any pre -treatments or the problems associated with the asexual reproduction of winterization to induce germination ( See Clarke , R C et al . cannabis . “ Cannabis : Evolution and Ethnobotany ” University of Cali [ 0335 ] This disclosure describes, inter alia , compositions, fornia Press 2013 ) . Seeds placed in viable growth conditions systems and methods for producing and maintaining Can are expected to germinate in about 3 to 7 days. The first true nabis cell explants, Cannabis tissue explants , Cannabis cell leaves of a cannabis plant contain a single leaflet, with cultures, isolated Cannabis cell cultures, Cannabis tissue subsequent leaves developing in opposite formation . In cultures, isolated Cannabis tissue cultures, Cannabis callus , some embodiments, subsequent leaves develop with increas and isolated Cannabis callus . In some embodiments , the ing number of leaflets . Leaflets can be narrow or broad compositions, systems and methods of the present invention depending on the morphology of the plant grown . Cannabis are used to produce clones of Cannabis plants, genotypes , plants are normally allowed to grow vegetatively for the first strains, and / or varieties . This can be accomplished , e.g. , via 4 to 8 weeks. During this period, the plant responds to regeneration of whole plants from the Cannabis tissue increasing light with faster and faster growth . Under ideal cultures produced according to the present invention . conditions , cannabis plants can grow up to 2.5 inches a day, [ 0336 ] The compositions, systems and methods of the and are capable of reaching heights of up to 20 feet. Indoor present invention can be used for the tissue culturing and growth pruning techniques tend to limit cannabis size plant regeneration of any Cannabis germplasm . With through careful pruning of apical or side shoots . twenty - six states and the District of Columbia in the United US 2020/0383331 A1 Dec. 10 , 2020 20
States legalizing marijuana in some form ( i.e. , for medical large numbers of resinous flowers, and thus typically exhibit and / or recreational use ) , Cannabis germplasms, strains, shorter, highly branched morphologies adapted to indoor varieties and /or lines are publicly and commercially avail grows. able . U.S. Pat . No. 6,630,507 issued on Oct. 7 , 2003 and [ 0340 ] Although marijuana cannabis stains as a drug and assigned on the patent face to The United States of America , industrial hemp both derive from the Cannabis family and is directed to methods of treating diseases caused by oxi contain the psychoactive component tetrahydrocannabinol dative stress by administering therapeutically effective ( THC ) , they are distinct strains with unique phytochemical amounts of a cannabidiol ( CBD ) cannabinoid from cannabis compositions and uses . Hemp has lower concentrations of plants that has substantially no binding to the N -methyl - D THC and higher concentrations of cannabidiol ( CBD ) , aspartate ( NMDA ) receptor, wherein the CBD acts as an which decreases or eliminates its psychoactive effects . The antioxidant and neuroprotectant. A search of the U.S.P.T.O legality of industrial hemp varies widely between countries. Patent Application Information Retrieval ( PAIR ) system Some governments regulate the concentration of THC and also reveals the existence of thousands of cannabis - related permit only hemp that is bred with an especially low THC applications and issued patents including U.S. Pat . No. content. 8,034,843 ( use of cannabinoids for treating nausea , vomit [ 0341 ] In contrast to cannabis for medical use , varieties ing , emesis, motion sickness ), U.S. Pat . No. 7,698,594 grown for fiber and seed have less than 0.3 % THC and are ( cannabinoid compositions for treatment of pain ) , and U.S. unsuitable for producing hashish and marijuana ( Sawler J et Pat . No. 8,632,825 ( anti- tumoural effects of cannabinoid al , 2015 , PLOS One . 10 ( 8 ) : e0133292 ) . Present in industrial combinations) among many others . Some examples of pub hemp , cannabidiolis a major constituent among some 560 licly - disclosed Cannabis germplasms, strains, varieties and / compounds found in hemp. The major differences between or lines each of which produce different amounts and / or the two types of plants are the appearance , and the amount ratios of cannabis metabolites can be found, e.g. , in U.S. Pat . of AP - tetrahydrocannabinol ( THC ) secreted in a resinous Nos . 9,095,554 ; 9,370,164 ; and 9,642,317 ; and U.S. Pub mixture by epidermal hairs called glandular trichomes, lished Patent Application Nos . 20110098348 ; 20140287068 ; although they can also be distinguished genetically. Oilseed 20160324091 ; and 20160360721 , each of which is specifi and fiber varieties of Cannabis approved for industrial hemp cally incorporated by reference herein in its entireties , production produce only minute amounts of this psychoac including all of the tables and figures. Specific strains of tive drug, not enough for any physical or psychological cannabis are disclosed in U.S. Published Patent Application effects . Typically, hemp contains below 0.3 % THC , while Nos . 20140245494 and 20160073567 ( ' Cannabis Plant cultivars of Cannabis grown for medicinal or recreational Named Erez ” ); 20140245495 20160073568 ( * Cannabis use can contain anywhere from 2 % to over 20 % . Plant Named Midnight' ); 20140259228 and 20160073566 [ 0342 ] Furthermore, President Obama signed the Agricul ( “ Cannabis Plant Named Avidekel ') ; 20160000843 ( * High tural Act of 2014 , or the 2014 Farm Bill , which included Cannabinol Cannabis Strains ' ); 20160345477 ( Cannabis Section 7606 allowing for universities and state departments Plant Named Ecuadorian Sativa ” ); and 20170172040 (“ Can of agriculture to begin cultivating industrial hemp for lim nabis Plant Named Katelyn Faith ' ) . ited purposes. Specifically, the law allows universities and state departments of agriculture to grow or cultivate indus [ 0337 ] In some embodiments, any medium , or combina trial hemp if: “ ( 1 ) the industrial hemp is grown or cultivated tions thereof, of the present disclosure may be utilized in for purposes of research conducted under an agricultural cannabis cultivation . pilot program or other agricultural or academic research ; and [ 0338 ] Also , the present invention teaches any medium , or ( 2 ) the growing or cultivating of industrial hemp is allowed combinations thereof, of the present disclosure may be under the laws of the state in which such institution of higher utilized in hemp culturing and / or cultivation . Hemp, or education or state department of agriculture is located and industrial hemp ( from Old English hcenep ), typically found such research occurs . ” For purposes of the Farm Bill , in the northern hemisphere , is a variety of the Cannabis industrial hemp is defined as Cannabis sativa L. , having a sativa plant species that is grown specifically for the indus THC concentration s0.3 % . trial uses of its derived products. is one of the fastest [ 0343 ] The law also requires that the grow sites be certi growing plants and was one of the first plants to be spun into fied by — and registered with — their state . A bipartisan group usable fiber 10,000 years ago . It can be refined into a variety of U.S. senators introduced the Industrial Hemp Farming of commercial items including paper , textiles, clothing , Act of 2015 that would allow American farmers to produce biodegradable plastics, paint, insulation , biofuel, food , and and cultivate industrial hemp . The bill would remove hemp animal feed . from the controlled substances list as long as it contained no [ 0339 ] Modern attempts at classifying cannabis now focus more than 0.3 percent THC . The U.S. Department of Agri on each individual plant's morphologies and intended use . culture, in consultation with the U.S. Drug Enforcement At the broadest level , cannabis plants can be categorized as Agency ( DEA ) and the U.S. Food and Drug Administration , either hemp or ‘ drug type' cannabis ( “ Marijuana ” ) strains. released a Statement of Principles on Industrial Hemp in the Hemp strains generally refer to fiber -producing cannabis Federal Register on Aug. 12 , 2016 , on the applicable activi plants that exhibit tall unbranched ( sativa - like ) morpholo ties related to hemp in the 2014 Farm Bill . gies . These plants have been bred to focus their energies on [ 0344 ] Hemp is used to make a variety of commercial and producing long fibrous stalks and generally only accumulate industrial products including rope , clothes , food , paper, low levels of cannabinoid drug compounds, which can be textiles , plastics , insulation and biofuel . The bast fibers can used for recreational or medicinal applications . “ Drug type ” be used to make textiles that are 100 % hemp , but they are cannabis ( “ Marijuana ” ) strains on the other hand, refer to commonly blended with other organic fibers such as flax , plants that are designed for human recreational or medicinal cotton or silk , to make woven fabrics for apparel and consumption. These plants focus their energies on producing furnishings. The inner two fibers of the plant are more US 2020/0383331 A1 Dec. 10 , 2020 21 woody and typically have industrial applications , such as salts , and agarose. Blends of these agents, such as two or mulch , animal bedding and litter . When oxidized ( often more of agar , carrageenan , gellan gum , agarose and alginic erroneously referred to as “ drying ” ), hemp oil from the seeds acid or a salt thereof also can be used . In some embodiments , becomes solid and can be used in the manufacture of no gelling agent or very little gelling agent is used for a oil - based paints, in creams as a moisturizing agent , for liquid medium . cooking , and in plastics . Hemp seeds have been used in bird [ 0352 ] In some embodiments , the media comprise one or feed mix as well . Also , more than 95 % of hemp seed sold in more minimum nutrition necessary for plant growth , such as the European Union was used in animal and bird feed amino acids , macroelements , microelements, aluminum , according to the 2013 research data . Thus, the hemp seed boron , chlorine ( chloride ) , chromium , cobalt , copper , iodine , can be used for animal and bird feed . iron , lead , magnesium , manganese , molybdenum , nitrogen [ 0345 ] In some embodiments, any medium , or combina ( nitrates ), potassium , phosphorous (phosphates ), silicon , tions thereof, of the present disclosure may be utilized in sodium , sulphur ( sulphates ), titanium , vanadium , zinc, inosi hemp culture and / or hemp cultivation . tol and undefined media components such as casein hydro lysates or yeast extracts . For example , the media can include Media any combination of NH4NO3; KNO3; Ca (NO3 ) 2; K2SO4 ; [ 0346 ] The present invention provides media comprising MgSO4; MnSO4; ZnSO4; K S0s; CuSO4; CaCl ; KI ; compounds with unique types, concentrations , and combi CoCl2 ; HZBO3 ; Na M004; KH , PO4; FeSO4; Na EDTA ; nations. In some embodiments, the medium is a liquid , Na H2PO4; inositol ( e.g. , myo - inositol ); thiamine ; pyridox semi- liquid , solid or semi - solid medium . ine ; nicotinic acid ; glycine ; and riboflavin . It is known to [ 0347 ] In some embodiments , liquid cultures offer several those in the art that one or more components mentioned advantages. The liquid cultivation saves time , because it above can be omitted without affecting the function of the enables replacement of the full medium in the vessel con media . taining multiple explants be made at once , instead of indi [ 0353 ] The media can comprise one or more carbon vidual transfers of single plant. In addition , a liquid culture source , such as a sugar . Non - limiting exemplary sugars results in increased shoot length because a larger area of the include sucrose , glucose , maltose , galactose and sorbitol or explant can get in contact with the medium . combinations thereof. [ 0348 ] The present invention provides media used for in [ 0354 ] In some embodiments, the media can comprise one vitro micropropagation of plants, such as bamboo plants and inorganic salts , growth regulators, carbon source , and / or agricultural plants . Media useful for the production of peren vitamins . In some embodiments , at least one of the vitamins nials , grasses and phyto -pharmaceutical plants , is also pro is provided by the Murashige and Skoog medium salts vided herein . (Murashige and Skoog , 1962 ) , Woody Plant ( WPM ) tissue [ 0349 ] Medium and methods used for plant micropropa culture salts , Driver Kuniyuki Walnut ( DKW ) tissue culture, gation have been described at least in M. R. Ahuja , Micro and / or functional variations thereof . propagation of woody plants, Springer, 1993 , ISBN [ 0355 ] The media further comprise one or more effective 0792318072 , 9780792318071 ; Narayanaswamy, Plant cell amount of plant growth regulators ( PGRs ) . Examples of and tissue culture , Tata McGraw - Hill Education, 1994 , plant growth regulators include plant hormones , such as ISBN 0074602772 , 9780074602775 ; Singh and Kumar, auxins and compounds with auxin - like activity, cytokinins Plant Tissue Culture , APH Publishing, 2009 , ISBN and compounds with cytokinin -like activity. The term " cyto 8131304396 , 9788131304396 ; Y. P. S. Bajaj High - tech and kinin ” refers to a class of plant growth regulators that are micropropagation V , Springer, 1997 , ISBN 3540616063, characterized by their ability to stimulate cell division and 9783540616061 ; Trigiano and Gray, Plant Tissue Culture , shoot organogenesis in tissue culture . Non - limiting Development and Biotechnology , CRC Press, 2010 , ISBN examples of cytokinins include, Nø -benzylaminopurine 1420083260 , 9781420083262 ; Gupta and Ibaraki, Plant ( BAP ) ( a.k.a. Nø -benzyladenine ( BA ) ) , meta - topolin , zeatin , tissue culture engineering Volume 6 of Focus on biotech kinetin , thiadiazuron ( TDZ ) , meta - topolin , 2 - isopentenylad nology , Springer, 2006 , ISBN 1402035942 , enine ( a.k.a. , 6 - y -v- (dimethylallylamino ) -purine or 2ip ) , 9781402035944 ; Jain and Ishii, Micropropagation ofwoody adenine hemisulfate , dimethylallyladenine, 4 - CPPU (N- ( 2 trees andfruits Volume 75 of Forestry sciences , Springer, chloro - 4 -pyridyl ) -N ' -phenylurea )) , and analogs thereof. The 2003 , ISBN 1402011350 , 9781402011351 ; and Aitken term “ auxin ” refers to a class of plant growth regulators that Christie et al . , Automation and environmental control in are characterized principally by their capacity to stimulate plant tissue culture , Springer, 1995 , ISBN 0792328418 , cell division in excised plant tissues . In addition to their role 9780792328414 , each of which is incorporated herein by in cell division and cell elongation , auxins influence other reference in its entirety . developmental processes , including root initiation . Non [ 0350 ] Medium and methods for bamboo micropropaga limiting examples of f3 -naphthoxyacetic acid (NAA ), 2,4 tion have been described in International Patent Application Dichlorophenoxy acetic acid ( 2,4 - D ) , indole - 3 -butyric acid Publication No. WO2011100762 , which is incorporated ( IBA ) , indole - 3 - acetic acid ( IAA ), picloram , and analogs herein by reference in its entirety . thereof. More cytokinins and auxins are described in [ 0351 ] The physical state of the media can vary by the WO2011100762 , U.S. Pat . No. 5,211,738 , US20100240537 , incorporation of one or more gelling agents. Any gelling US20060084577 , US20030158043 , and Aremu et al . , 2011 , agent known in the art that is suitable for use in plant tissue which are incorporated by reference in their entireties . In culture media can be used . Agar is most commonly used for some embodiments , the cytokinin is BAP or any functional this purpose . Examples of such agars include Agar Type A , variant thereof. In some embodiments , the auxin is IAA or E or M and BactoTM Agar. Other exemplary gelling agents any functional variant thereof. include carrageenan , gellan gum ( commercially available as [ 0356 ] In some embodiments, other plant growth regula PhytaGelTM , Gelrite® and GelzanTM ), alginic acid and its tors can be added in the media to improve cell growth and US 2020/0383331 A1 Dec. 10 , 2020 22 development. In some embodiments , growth inhibitors and / [ 0361 ] Optionally , the media further comprise one or more or growth retardants are used . buffering agent. The buffering agent can buffer the salt [ 0357 ] Non - limiting examples of growth inhibitors concentration and / or the pH in the medium . For example, include , abscisic acid , ancymidol, butralin , carbaryl, chlo the buffering agent can maintain the pH of the liquid mixture rphonium , chlorpropham , dikegulac , flumetralin , fluorid so the pH is kept around about 4.0 , about 4.1 , about 4.2 , amid , fosamine, glyphosine, isopyrimol, jasmonic acid , about 4.3 , about 4.4 , about 4.5 , about 4.6 , about 4.7 , about maleic hydrazide, mepiquat, piproctanyl, prohydrojasmon , 4.8 , about 4.9 , about 5.0 , about 5.1 , about 5.2 , about 5.3 , propham , tiaojiean , and 2,3,5 - tri - iodobenzoic acid , deriva about 5.4 , about 5.5 , about 5.6 , about 5.7 , about 5.8 , about tives thereof, or combinations thereof. 5.9 , about 6.0 , about 6.1 , about 6.2 , about 6.3 , about 6.4 , [ 0358 ) Non - limiting examples of growth retardant about 6.5 about 6.6 , about 6.7 , about 6.8 , about 6.9 , or about include , ancymidol ( e.g. , A - Rest® , Abide® ), chlormequat 7.0 . For example, the pH of the liquid mixture is in a range chloride ( e.g. , Chlormequat E - Pro® , Citadel® , Cycocel® ) , between about 5.0 to about 7.0 . In some embodiments, the daminozide ( e.g. , B - Nine® , Dazide® ) , ethephon ( e.g. , Flo buffering agent is 2- ( N -morpholino ) ethanesulfonic acid rel® ) , flurprimidol ( e.g. , Topflor® ), paclobutrazol ( e.g. , ( MES ) , Adenosine deaminase ( ADA ), piperazine - N , N ' - bis Bonzi , Downsizer , Paczol® , Piccolo ) , mefluidide, ( 2 - ethanesulfonic acid ) ( PIPES ) , N-( 2 - Acetamido )-2 -ami paclobutrazol, tetcyclacis and uniconazole ( e.g. , Conciser , noethanesulfonic acid ( ACES ) , Cholamine chloride , etc. In Sumagic® ) . In some embodiments , the growth retardant is some embodiments, the buffering agent is IViES, and its an gibberellic acid (GA3 ) antagonist which can inhibit GA3 concentration is about 500-1200 mg / L . In some embodi pathway, for example , ancymidol , tannins, paclobutrazol ments , the pH of the medium is maintained at about 5.5 to ( PBZ ), ( 2 -Chloroethyl ) trimethylammonium chloride , absci 6.5 , for example , about 5.8 . In some embodiments, the pH sin , exogenous ABA , derivatives thereof, or combinations of the medium is maintained at about 5.0 to 6.0 , for example , thereof. about 5.7 . [ 0359 ] Exemplary concentrations of the components [ 0362 ] If present in a media , each cytokinin can be present described above are shown in Table 1. The concentrations of in an amount from about 0.001 mg / L - about 10 mg / L and all these components can be adjusted based on plant species , amounts in between . For example, the concentration of a tissue type, and purposes , etc , without substantially affecting cytokinin is about 0.001 , 0.01 , 0.1 , 1 , 2 , 3 , 3.1 , 3.2 , 3.3 , 3.4 , the media function . The exemplary concentrations are by no 3.5 , 3.6 , 3.7 , 3.8 , 3.9 , 4 , 4.1 , 4.2 , 4.3 , 4.4 , 4.5 , 4.6 , 4.7 , 4.8 , means limiting , and merely encompass some of the embodi 4.9 , 5 , 5.1 , 5.2 , 5.3 , 5.4 , 5.5 , 5.6 , 5.7 , 5.8 , 5.9 , 6 , 6.1,6.2 , 6.3 , ments . 6.4 , 6.5 , 6.6 , 6.7 , 6.8 , 6.9 , 7 , 7.1 , 7.2 , 7.3 , 7.4 , 7.5 , 7.6 , 7.7 , 7.8 , 7.9 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , TABLE 1 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , Exemplary Concentrations 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , Concentrations 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , (mg / L in all unless 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99. or about Component otherwise noted ) 100 mg / L . In some embodiment, at least one cytokinin is meta - topolin , and its concentration is about 0.2 to 20 mg / L , NH4NO3 about 800 - about 2500 KNO3 about 900 - about 3000 for example , about 2 mg/ L . In some embodiment, at least Ca (NO3 ) 2 0 - about 800 one cytokinin is 2ip , and its concentration is about 1 to 10 K2SO4 0 - about 800 mg / L , for example, about 4-5 mg / L . MgSO4 about 150 - about 550 MnSO4 about 8.0 - about 26.0 [ 0363 ] In some embodiments, the media comprise meta ZnSO4 about 4.0 - about 12.0 topolin and / or its analogues. In some embodiments , meta CuSO4 about 0.010 - about 0.4 topolin is present in an amount equal to or greater than 1.5 CaCl2 about 200 - about 660 mg / L , equal to or greater than 2.0 mg/ L , equal to or greater KI about 0.4 - about 1.5 CoCl2 about 0.010 - about 0.4 than 2.5 mg / L , equal to or greater than 3.0 mg / L , equal to or HZBO3 about 3.0 - about 9.0 greater than 3.5 mg / L , equal to or greater than 4.5 mg / L or Na M004 about 0.10 - about 0.4 equal to or greater than 5.0 mg/ L . In other embodiments , KH2PO4 about 80 - about 250 meta - topolin is present in an amount of 3.2 mg / L or 5.36 FeSO4 about 25 - about 90 Na EDTA about 35 - about 120 mg / L . In another embodiment, the amount of meta - topolin Na2H2PO4 about 0-250 / about 80-250 cannot be less than 1.5 mg / L , cannot be less than 2.0 mg / L , myo - Inositol about 50 - about 150 cannot be less than 2.5 mg / L , cannot be less than 3.0 mg / L , Thiamine about 0.2 - about 0.6 cannot be less than 3.5 mg / L , cannot be less than 4.5 mg / L Pyridoxine about 0.1 - about 10 Nicotinic acid about 0.1 - about 10 or cannot be less than 5.0 mg / L . In some embodiments , Sugar about 10 g / L - about 100 g / L meta - topolin and / or its analogues can be included in any Glycine about 0 - about 5 amount up to 200 mg/ L . In some embodiments , the media is Riboflavin about O - about 5 used for bamboo micropropagation . In some embodiments , Ascorbic Acid about 0 - about 5 the bamboo plant is selected from the species consisting of Gelling agent * about 2.5 g / L - about 8.0 g / L Arundinaria, Bambusa, Borinda, chusquea, Dendrocalamus, * The amount of gelling agent may vary depending on the type of the agent , and the type fargesia, Guadua , Phyllostachys, Pleioblastus and Thamno of the media ( e.g., semi- solid or solid media ) calamus . [ 0360 ] As used herein and in the claims , where the term [ 0364 ] In some embodiments, the media comprise thidi “ about " is used with a numerical value , the numerical value azuron and / or its analogues. In some embodiments , thidiaz may vary from the explicit number ; the variation will be uron and / or its analogues can be present at 0.001 mg / L , 0.01 , + 10 % . 0.025 , 0.05 , 0.075 , 0.1 , 0.15 , 0.2 , 0.25 , 0.3 , 0.35 , 0.4 , 0.45 , US 2020/0383331 A1 Dec. 10 , 2020 23
0.5 , 0.55 , 0.6 , 0.65 , 0.7 , 0.75 , 0.8 , 0.85 , 0.9 , 0.95 , 1.0 , 1.25 , ments, the sucrose concentration in the propagation and 1.50 , 1.75 , 2.0 , 2.25 , 2.5 , 2.75 , 3.5 , 4.5 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , multiplication media is about 20-40 g / L or higher. 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , [ 0370 ] In some embodiments, at least one auxin is mT . In 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , some embodiments , at least one cytokinin is IBA . In some 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , embodiments , the media comprise at least one gelling agent 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , such as agar. In some embodiments, the media comprise at 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , least one gelling agent such as agar. In some embodiments , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 or 100 mg / L . In some the concentration of the gelling agent is about 4 to about 10 embodiments, thidiazuron and /or its analogues can also be grams, for example, about 5 grams. included in any amount up to 200 mg / L . [ 0371 ] In some embodiments, the initiation medium and / [ 0365 ] If present in a media , each auxin can be present in or the multiplication medium comprise MS medium con an amount from about 0.01 mg/ L - about 100 mg / L and all taining double concentration of meso elements ( e.g. , one or amounts in between . For example , the concentration of an more of CaC12.2H2O , MgSO4.7H2O , and KH2PO4 ) , auxin is about 0.001 , 0.01 , 0.02 , 0.03 , 0.04 , 0.05 , 0.06 , 0.07 , double iron , and one or more Gamborg's vitamins ( e.g. , one 0.08 , 0.09 , 0.1 , 0.5 , 1 , 2 , 3 , 3.1 , 3.2 , 3.3 , 3.4 , 3.5 , 3.6 , 3.7 , or more of myo - inositol, Nictotinic acid , pyridoxine salts , 3.8 , 3.9 , 4 , 4.1 , 4.2 , 4.3 , 4.4 , 4.5 , 4.6 , 4.7 , 4.8 , 4.9 , 5 , 5.1 , 5.2 , and thiamine salts ) . 5.3 , 5.4 , 5.5 , 5.6 , 5.7 , 5.8 , 5.9 , 6 , 6.1 , 6.2 , 6.3 , 6.4 , 6.5 , 6.6 , [ 0372 ] The third type of media , referred herein as the 6.7 , 6.8 , 6.9 , 7 , 7.1 , 7.2 , 7.3 , 7.4 , 7.5 , 7.6 , 7.7 , 7.8 , 7.9 , 8 , 9 , rooting media , are similar to or essentially the same as the 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , Murashige and Skoog medium , Woody Plant ( WPM ) tissue 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , culture salts , Driver Kuniyuki Walnut ( DKW ) , but comprise 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , at least one auxin . In some embodiments, the auxin is IBA . 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , In some embodiments, the media do not comprise any 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , cytokinin . In some embodiments, the sucrose concentration 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 or about 100 mg / L . In in the propagation and multiplication media is about 20-40 some embodiments, at least one auxin is IAA , and the g / L or at least 60 g / L . In some embodiments, the concen concentration is about 0.1 to 10 mg / L , for example, about 1 tration of IBA is about 0.1 to 10 mg / L . In some embodi mg / L . In some embodiments, at least one auxin is NAA , and ments , the concentration of IBA is about 100 to 1500 mg / L . the concentration is about 0.001 to 1 mg / L , for example , In some embodiments, the concentration of the gelling agent about 0.02 mg/ L . is about 4 to about 10 grams, for example, about 6 grams. [ 0366 ] In some embodiments , at least one auxin is NAA , [ 0373 ] In some embodiments , the micropropagation and IBA or combination thereof. In some embodiments, at least multiplication media ( or elongation and multiplication one auxin is IBA . In some embodiments , NAA or IBA is media ), is similar to or essentially the same as the Murashige presented in an initiation medium or a multiplication and Skoog medium (Murashige and Skoog, 1962 ) , but medium , and the concentration is about 0.01-10 mg / L , for without any hormone or growth regulator. In some embodi example , about 0.02-1 mg / L . In some embodiments, NAA or ments, the sucrose concentration in the propagation and IBA is presented in a rooting medium . In some embodi multiplication media is about 10-20 g / L . ments , the NAA /MA concentration in a rooting medium is [ 0374 ] In some embodiments, the pre - tuberization media , about 1 to 10 mg / L , for example, about 1-3 mg / L . In some comprises one or more cytokinins and one or more auxin . In embodiments, the NAA / IBA concentration in a rooting some embodiments, the cytokinin is 2ip and the auxin is medium is about 100-1500 mg / L , for example , about 250 IAA . Alternatively, instead of cytokinin and auxin , the 1000 mg / L . pre -tuberization media comprises one or more plant retar [ 0367 ] In some embodiments, the present invention pro dant in low amount, such as ancymidol or analog thereof. In vides several types of media that are used in in vitro some embodiments , the pre -tuberization media comprise micropropagation of plants of the present disclosure . one or more cytokinin , one or more auxin , and one or more [ 0368 ] The first type of media, referred herein as the growth retardant. In some embodiments , the concentration initiation media, is similar to or essentially the same as the of 2ip is about 1 to 10 mg/ L , for example, about 4-5 mg / L . Murashige and Skoog medium (Murashige and Skoog , In some embodiments , the concentration of IAA is about 0.1 1962 ) , Woody Plant ( WPM ) tissue culture salts , Driver to 10 mg / L , for example, about 1 mg/ L . In some embodi Kuniyuki Walnut ( DKW ) , but comprise at least one auxin ments, the concentration of ancymidol is about 0.1 to 10 and / or at least one cytokinin . In some embodiments , the mg / L . In any case , the sucrose concentration is about 20 g / L sucrose concentration in the propagation and multiplication to about 40 g / L , for example , about 30 g / L . media is about 20-40 g / L or higher. In some embodiments , [ 0375 ] In some embodiments, the tuberization media , at least one auxin is mT . In some embodiments, at least one comprises one or more auxin , but does not comprise any cytokinin is NAA . In some embodiments , the media com cytokinin . Alternatively , instead of auxin , the tuberization prise at least one gelling agent such as agar . In some media comprises one or more plant retardant, such as embodiments , the concentration of the gelling agent is about ancymidol or analog thereof. In some embodiments, the 4 to about 10 grams, for example, about 7 grams. In some tuberization media comprise one or more auxin and one or embodiments , the initiation media do not contain more growth retardant. In some embodiments, the auxin is Na2H2PO4 . In some embodiments, the initiation media do NAA . In some embodiments, the NAA concentration is not contain pyridoxine , nicotinic acid , and / or riboflavin . about 0.01 to about 0.05 mg / L . In some embodiments, the [ 0369 ] The second type of media , referred herein as the concentration of ancymidol is about 0.1 to 10 mg / L . In any micropropagation media or multiplication media , are as the case , the sucrose concentration in the media is higher same as , or similar to the initiation media . In some embodi compared to the sucrose concentration in the pre -tuberiza US 2020/0383331 A1 Dec. 10 , 2020 24 tion media . For examples, the sucrose concentration in the 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 or 100 mg / L . In some tuberization media is about 50 g / L to about 100 g / L or more . embodiments, 2ip and / or its analogues can be included any [ 0376 ] In some embodiments, the media comprise NAA amount up to 200 mg/ L . and / or its analogues . In some embodiments , NAA and / or its [ 0380 ] In some embodiments, the media comprise DPU analogues can be present at 0.001 mg / L , 0.01 , 0.0125 , 0.015 , and / or its analogues . In some embodiments, DPU and / or its O.Ol75,0.2, 0.025 0.025 0.0275 0.03 0.035, 0.035 , analogues can be present at 0.001 mg / L , 0.01 , 0.025 , 0.05 , 0.0375 , 0.04 , 0.0425 , 0.045 , 0.0475 , 0.05 , 0.0525 , 0.055 , 0.075 , 0.1 , 0.15,0.2 , 0.25 , 0.3 , 0.35 , 0.4 , 0.45 , 0.5 , 0.55,0.6 , 0.0575 , 0.06 , 0.0625 , 0.065 , 0.0675 , 0.07 , 0.0725 , 0.075 , 0.65 , 0.7 , 0.75 , 0.8 , 0.85 , 0.9 , 0.95 , 1.0 , 1.25 , 1.50 , 1.75 , 2.0 , 0.0775 , 0.08 , 0.0825 , 0.085 , 0.0875 , 0.09 , 0.0925 , 0.095 , 2.25 , 2.5 , 2.75 , 3.5 , 4.5 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 0.0957 , 0.1 , 0.15 , 0.2 , 0.25 , 0.3 , 0.35 , 0.4 , 0.45 , 0.5 , 0.55 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 0.6 , 0.65 , 0.7 , 0.75 , 0.8 , 0.85 , 0.9 , 0.95 , 1.0 , 1.25 , 1.50 , 1.75 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 2.0 , 2.25 , 2.5 , 2.75 , 3.5 , 4.5 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 96 , 97 , 98 , 99 or 100 mg / L . In some embodiments, DPU 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , and /or its analogues can be included any amount up to 200 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , mg / L . 95 , 96 , 97 , 98 , 99 or 100 mg / L . In some embodiments, NAA [ 0381 ] In some embodiments , the media comprise CPPU and / or its analogues can be included any amount up to 200 and / or its analogues. In some embodiments, CPPU and / or its mg / L . analogues can be present at 0.001 mg / L , 0.01 , 0.025 , 0.05 , [ 0377 ] In some embodiments, the media comprise IBA 0.075 , 0.1 , 0.15,0.2 , 0.25 , 0.3 , 0.35 , 0.4 , 0.45 , 0.5 , 0.55,0.6 , and / or its analogues. In some embodiments, IBA and / or its 0.65 , 0.7 , 0.75 , 0.8 , 0.85 , 0.9 , 0.95 , 1.0 , 1.25 , 1.50 , 1.75 , 2.0 , analogues can be present at 0.001 mg / L , 0.01 , 0.025 , 0.05 , 2.25 , 2.5 , 2.75 , 3.5 , 4.5 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 0.075 , 0.08 , 0.1 , 0.125 , 0.15 , 0.175 , 0.2 , 0.225 , 0.25 , 0.275 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 0.3 , 0.325 , 0.35 , 0.375 , 0.4 , 0.425 , 0.45 , 0.475 , 0.5 , 0.525 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 0.55 , 0.575 , 0.6 , 0.625 , 0.65 , 0.675 , 0.7 , 0.725 , 0.75 , 0.775 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 0.8 , 0.825 , 0.85 , 0.875 , 0.9 , 0.925 , 0.95 , 0.975 , 1.0 , 1.25 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 1.50 , 1.75 , 2.0 , 2.25 , 2.5 , 2.75 , 3.0 , 3.25 , 3.5 , 3.75 , 4.0 , 4.5 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 96 , 97 , 98 , 99 or 100 mg / L . In some embodiments, CPPU 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , and / or its analogues can be included any amount up to 200 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , mg / L . 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , [ 0382 ] In some embodiments, one or more cytokinins in 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 or 100 combination with one or more other cytokinins or auxins, mg / L . In some embodiments, IBA and / or its analogues can and auxins in combination with other auxins or cytokinins be included any amount up to 200 mg / L . can also be utilized in ratios. For example , in some embodi ments, any two cytokinins and / or auxins in pairs disclosed [ 0378 ] In some embodiments, the media comprise BAP herein can be included in the following exemplary ratios : and / or its analogues . In some embodiments, BAP and / or its 100 : 1 , 95 : 1 , 90 : 1 , 85 : 1 , 80 : 1 , 75 : 1 , 70 : 1 , 65 : 1 , 60 : 1 , 55 : 1 , analogues can be present at 0.001 mg/ L , 0.01 , 0.025 , 0.05 , 50 : 1 , 45 : 1 , 40 : 1 , 35 : 1 , 30 : 1 , 29 : 1 , 28 : 1 , 27 : 1 , 26 : 1 , 25 : 1 , 0.06 , 0.07 , 0.0725 , 0.075 , 0.0775 , 0.08 , 0.0825 , 0.085 , 24 : 1 , 23 : 1 , 22 : 1 , 21 : 1 , 20 : 1 , 19 : 1 , 18 : 1 , 17 : 1 , 16 : 1 , 15 : 1 , 0.0875 , 0.09 , 0.0925 , 0.095 , 0.0975 , 0.1 , 0.125 , 0.15 , 0.175 , 14 : 1 , 13 : 1 , 12 : 1 , 11 : 1 , 10 : 1 ; 9 : 1 , 8 : 1 , 7 : 1 , 6.9 : 1 , 6.8 : 1 , 6.7 : 1 , 0.2 , 0.225 , 0.25 , 0.275 , 0.3 , 0.325 , 0.35 , 0.375 , 0.4 , 0.425 , 6.6 : 1 , 6.5 : 1 , 6.4 : 1 , 6.3 : 1 , 6.2 : 1 , 6.1 : 1 , 6 : 1 , 5.9 : 1,5.8 : 1 , 5.7 : 1 , 0.45 , 0.475 , 0.5 , 0.525 , 0.55 , 0.575 , 0.6 , 0.625 , 0.65 , 0.675 , 5.6 : 1 , 5.5 : 1 , 5.4 : 1 , 5.3 : 1 , 5.2 : 1 , 5.1 : 1 , 5 : 1 ; 4 : 1 , 3 : 1 , 2 : 1 , 1 : 1 , 0.7 , 0.75 , 0.8 , 0.85,0.9 , 0.95 , 1.0 , 1.25 , 1.50 , 1.75 , 2.0 , 2.25 , 0.75 : 1 , 0.5 : 1 , 0.25 : 1 , 0.1 : 1 , 0.075 : 1 , 0.05 : 1 , 0.025 : 1 or 2.5 , 2.75 , 3.5 , 4.5 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 0.001 : 1 . These ratios can also be utilized between meta 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , topolin ( and analogues ) with thidiazuron ( and analogues ), 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , with NAA ( and analogues ), with BAP ( and analogues ), with 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , IBA ( and analogues ), with Zip ( and analogues ) , with DPU 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , ( and analogues ) and / or with CPPU ( and analogues ) . Simi 82 , 83 , 84 , 85 , 86 , 87 , 88,899,99,92,93,94,95,96,97, larly, the ratios can be utilized between thidiazuron ( and 98 , 99 or 100 mg/ L . In some embodiments , BAP and /or its analogues) with NAA ( and analogues ), with BAP ( and analogues can be included any amount up to 200 mg / L . analogues ), with IBA ( and analogues ), with 2ip ( and ana [ 0379 ] In some embodiments , the media comprise 2ip logues ) , with DPU ( and analogues ) and / or with CPPU ( and and / or its analogues . In some embodiments, 2ip and / or its analogues ). The ratios can also be utilized between NAA analogues can be present at 0.001 mg / L , 0.01 , 0.025 , 0.05 , ( and analogues ) with BAP ( and analogues ) , with IBA ( and Q.Q75,0.98 , Q.1 , Q.150.20.25 , Q.3 , 0.3 , 0.4 , Q.4 , Q.5 , analogues ), with 2ip ( and analogues ) , with DPU ( and ana 0.55,0.6 , 0.65 , 0.7 , 0.75 , 0.8 , 0.85 , 0.9 , 0.95 , 1.0 , 1.25 , 1.50 , logues ) and /or with CPPU ( and analogues ). The ratios can 1.75 , 2.0 , 2.25 , 2.5 , 2.75 , 3.0 , 3.5 , 4 , 0 , 4.5 , 5 , 6 , 7 , 8 , 9 , 10 , also be utilized between BAP ( and analogues ) with IBA ( and 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , analogues ), with 2ip ( and analogues ) , with DPU ( and ana 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , logues ) and / or with CPPU ( and analogues ) . In short, each of 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , the cytokinins and / or auxins or its analogues can be included 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , with a second cytokinin and /or auxin disclosed herein 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , according to any of the disclosed ratios . US 2020/0383331 A1 Dec. 10 , 2020 25
[ 0383 ] In some embodiments, the present invention pro seed incubated in media that is not BOO32 , BOO33 , or vides different types of media that are useful in the produc BOO34 . Phenolic production can be determined by HPLC tion of perennials , grasses and phyto - pharmaceutical plants. DAD ( diode array detector ), LC - MS / MS , or any other In some embodiments, the medium useful for producing method known in the arts . In some embodiments , the perennials , grasses and phyto - pharmaceutical plants is a medium useful for reducing phenolic production in bamboo liquid medium . In some embodiments, the medium is a solid is a liquid medium . In some embodiments, the medium is a medium . solid medium . [ 0384 ] The media useful for the production of perennials, [ 0388 ] In some embodiments, the present invention pro grasses , and phtyo -pharmaceutical plants can be any one of vides different types of media that are useful in the produc the media described in FIGS . 26A , 26B , and 27. In one tion of virus - free plants, such as agricultural plants. The embodiment, the media is selected from FIGS . 26A and 26B . media useful for the production of virus - free plants can In one embodiment, the media is Pulsing media 1 or Pulsing comprise an antiviral. The antiviral can be acemannan , media 2 from FIGS . 26A and 26B . In another embodiment, acyclovir, adefovir, alovudine , alvircept, amantadine, ara the media is Pulsing media 1 , wherein TDZ is substituted notin , arildone , atevirdine, pyridine , cidofovir , cipamfylline , with a different cytokinin, such as BAP, Zeatin , CPPU , or cytarabine, desciclovir, disoxaril , edoxudine , enviradene , DPU . In another embodiment, the media is B0068 , BO069 , enviroxime, famdclovir, famotine, fiacitabine, fialuridine , B0070 , or B0071 from FIG . 27. In some embodiments , floxuridine, fosarilate , fosfonet, ganciclovir, idoxuridine , B0068 , BO069 , BOO70 , B0071 are used as rooting kethoxal, lobucavir, memotine , methisazone, penciclovir, media . In yet another embodiment, the media is BO054 , pirodavir, somantadine , sorivudine, tilorone, trifluridine, BO055 , BO056 , BO057 , BO058 , BO059 , B0060 , valaciclovir , vidarabine, viroxime , zinviroxime, moroxy BO061 , BOO62 , BO063 , BOO64 , BO065 , BO066 , or dine , podophyllotoxin , ribavirine, rimantadine, stallimycine , BOO67 media from FIGS . 26A and 26B . statolon , tromantadine and xenazoic acid , and their pharma [ 0385 ] In some embodiments , a combination of the media ceutically acceptable salts . In one embodiment, the antiviral described in FIGS . 26A and 26B is useful for the production is ribavirine ( also known as Virazole ) or derivatives thereof, of perennials, grasses, and phyto - pharmaceutical plants. The such as viramidine ( also known as Taribavirin ). In some combination of media can be used sequentially . In one embodiments, a media useful for the production of virus embodiment, the combination comprises Pulsing media 1 , free plants comprises one or more antivirals. Pulsing media 2 or both ; and BO054 , BO055 , BO056 , BOO57 , BO058 , BO059 , B0060 , BO061 , B0062 , [ 0389 ] The media useful for the production of virus - free B0063 , B0064 , BO065 , BO066 , or B0067 media from plants can be any one of the media described in FIG . 32. In FIGS . 26A and 26B . In some embodiments, the combination one embodiment, the media is BOO81 . In some embodi comprises Pulsing media 1 , Pulsing media 2 or both ; and ments , a combination of the media described in FIG . 32 is B0068 , BOO69 , B0070 , or B0071 from FIG . 27. In some useful for the production of virus - free plants . The combi embodiments , the combination comprises Pulsing media 1 , nation of media can be used sequentially. In one embodi Pulsing media 2 or both ; BO054 , BO055 , B0056 , B0057 , ment, the combination comprises BOO81 and a regeneration BO058 , BO059 , B0060 , B0061 , B0062 , BO063 , media . In some embodiments, the combination comprises B0064 , B0065 , B0066 , or BO067 media from FIG . 27 ; BOO81 and at least two or more regeneration media . and B0068 , B0069 , BOO70 , or B0071 from FIG . 27. In [ 0390 ] A regeneration media can promote the growth of some embodiments, the Pulsing media 1 in the combination explants ( such as explants incubated in a medium with an is Pulsing media 1 , wherein TDZ is substituted with a antiviral, such as BOO81 ) and apical meristems ( such as different cytokinin , such as BAP , Zeatin , CPPU , or DPU . apical meristems from explants incubated in a medium with [ 0386 ] In some embodiments, wasabi is grown in B0051 , an antiviral) . In some embodiments, the regeneration media lily is grown in B0052 ( light) and / or B0053 ( dark ), aloe comprises an antiviral. In other embodiments, the regenera vera is grown in BOO54 , ginger is grown in B0055 , grape tion media does not comprise an antiviral. The regeneration is grown in B0056 , cannabis is grown in B0057 , garlic is can be BOO82 , BO083 , BOO84 , BO085 , BO086 , BO087 , grown in B0058 , onion is grown in B0059 hakonechloa is BO088 , BO089 , BOO90 , or BO091 , as depicted in FIG . grown in B0060 , miscanthus is grown in B0061 , Arundo 32 . donax is grown in B0062 , switch grass is grown in B0063 , [ 0391 ] In one embodiment, the combination of media rice is grown in B0064 , sugar cane is grown in B0065 , useful for the production of virus - free plants comprises echinacea is grown in B0066 , and / or geranium is grown in BOO81 , BOO82 and BOO83 . In another embodiment, the BOO67 . combination comprises BOO81 and BOO84 . In another [ 0387 ] In some embodiments, the present invention pro embodiment, the combination comprises B0081 , BO085 , vides different types of media that are useful in the reduction and B0086 . In yet another embodiment, the combination of phenolic production in plants, such as from bamboos. The comprises BOO81 , BO087 and BO088 . In yet another media useful for induction of somatic embryos can be any embodiment, the combination comprises BOO81 and one of the media described in FIG . 20 ( i.e. BOO32 , BO033 , BOO89 . In yet another embodiment, the combination com BO034 ) . The media can be useful in reducing the produc prises BOO81 and Sugar Beeet . In yet another embodiment, tion of a phenolic, such as a polyphenol, in bamboo . In some the combination comprises B0081 and B0091 . In some embodiments, the phenolic is a luteolin derivative, flavone , embodiments BOO82 and / or BO083 may be used with flavone glycoside or phenolic acid . The reduction of phe potato , BOO84 may be used with tomato , BOO85 and /or nolic production can be determined by comparing the phe B0086 may be used with cucumber, BO087 and / or B0088 nolic production of a bamboo tissue culture , explants or seed may be used with yam , B0089 may be used with cauli incubated in BOO32 , BOO33 , or BOO34 media to the flower, BOO90 may be used with sugar beet , BO091 may phenolic production of a bamboo tissue culture , explants or be used with cassava . US 2020/0383331 A1 Dec. 10 , 2020 26
[ 0392 ] In some embodiments, the medium useful for pro grasses and phyto -pharmaceutical plants , is about 0.25 mg / L ducing virus - free plants is a liquid medium . In some to about 100 mg / L , for example, about 0.5 mg / L to about 2 embodiments, the medium is a solid medium . mg / L . Thus, the concentration of TDZ or analog thereof can , [ 0393 ] In other embodiments, the present invention pro for example , be about 0.25 mg/ L , about 0.3 mg / L , about 0.4 vides at least two types of media that are used in in vitro mg / L , about 0.5 mg / L , about 0.6 mg / L , about 0.7 mg / L , micropropagation . The first media , referred herein as the about 0.8 mg / L , about 0.9 mg / L , about 1.0 mg/ L , about out “ bud induction media ( B001 ) ” , comprises at least one strong 1.5 mg/ L , about 2.0 mg / L , about 2.5 mg / L , about 3 mg / L , cytokinin , such as a thidiazuron , or analogs thereof . The about 4 mg / L , about 5 mg / L , about 6 mg / L , about 7 mg / L , second media , referenced herein as the “ shoot elongation / about 8 mg / L , about 9 mg / L , about 10 mg / L , about 15 mg / L , maintenance media ( BOO2 ) ” , comprises one or more cyto about 20 mg / L , about 25 mg / L , about 30 mg / L , about 40 kinins other than the cytokinin in the bud induction media mg / L , about 50 mg/ L , about 60 mg / L , about 70 mg / L , about ( B001 ) . For example, the cytokinins are selected from 80 mg / L , about 90 mg / L or about 100 mg / L . meta - topolin , kinetin , isopentenyl adenine ( iP ) , zeatin , trans [ 0399 ] In some embodiments , the bud induction media zeatin , zeatin riboside, dihydrozeatin , benzyleadenin ( BAP ) , ( BO01 ) comprises only one strong cytokinin , for example, benzyladenosine ( [ 9R ] BAP ) , analogs thereof, or combina thidiazuron ( TDZ ) , or an analog thereof. In some embodi tion thereof. Other cytokinins available for use in tissue ments, the embryo induction media or bud induction media culture can also be substituted for the above cytokinins to ( B001 ) comprises one additional cytokinin . In some achieve similar effects . embodiments, the embryo induction media or bud induction [ 0394 ] In some embodiments, the bud induction medium media ( B001 ) further comprises one or more auxin , such as ( B001 ) is a liquid medium . In some embodiments , the bud NAA , 2,4 - D , IBA , IAA , picloram , or analogs thereof. induction medium ( B001 ) is a solid medium . In some [ 0400 ] In some embodiments, the concentration of the embodiments, the shoot elongation /maintenance medium strong cytokinin ( e.g. , TDZ ) in the bud induction media ( BOO2 ) is a liquid medium . In some embodiments, the ( B001 ) is about 0.25 mg / L ( + 10 % ) to about 100 mg / L shoot elongation /maintenance media ( B002 ) is a solid ( + 10 % ) , for example, is about 0.2 mg / L ( + 10 % ) , about 0.5 media . mg / L ( + 10 % ) , about 1.0 mg / L ( + 10 % ) , about 5 mg / L [ 0395 ] The bud induction media ( B001 ) , the shoot elon ( + 10 % ) , about 10 mg / L ( + 10 % ) , about 20 mg / L ( + 10 % ) , gation /maintenance media ( B002 ) , and media useful for about 30 mg/ L ( + 10 % ) , about 40 mg / L ( + 10 % ) , about 50 producing perennials, grasses and phyto -pharmaceutical mg / L ( + 10 % ) , about 60 mg/ L ( + 10 % ) , about 70 mg / L plants comprise components of a minimum media for plant ( + 10 % ) , about 80 mg / L ( + 10 % ) , about 90 mg/ L ( + 10 % ) , or tissue culture , such as carbon source and salts . In some about 100 mg / L ( + 10 % ) . For example , the concentration of embodiments , the media can comprise one or more compo TDZ is about 0.2 ( -10 % ) to about 20 ( -10 % ) mg/ L , about nents selected from NH4NO3, KNO3, Ca (NO3 ) 2, K , SO4, 0.4 ( 10 % ) to about 10 ( -10 % ) mg / L , or about 0.5 ( + 10 % ) MgSO4 , MnSO4, ZnSO4 , CuSO4 , K2S05 , CaCl2 , K1 , CoCl2 , to about 2 ( + 10 % ) mg/ L . HZBO3 , Na M004, KH2PO4 FeSO4, Na EDTA , [ 0401 ] In some embodiments , the concentration of the Na H2PO4 , Glycine , myo - Inositol, Thiamine , Pyridoxine, strong cytokinin ( e.g. , TDZ ) in the media useful for produc Nicotinic acid , and Riboflavin . ing virus - free plants, such as in agricultural plants , is about [ 0396 ] In some embodiments , the media useful for pro 0.25 mg / L ( + 10 % ) to about 100 mg / L ( + 10 % ) , for example, ducing perennials, grasses and phyto - pharmaceutical plants is about 0.2 mg / L ( + 10 % ) , about 0.5 mg / L ( + 10 % ) , about 1.0 comprises only one strong cytokinin, for example, thidiaz mg/ L ( + 10 % ), about 5 mg / L ( + 10 % ) , about 10 mg / L uron ( TDZ ) , or an analog thereof. In some embodiments , the ( + 10 % ) , about 20 mg / L ( + 10 % ) , about 30 mg / L ( + 10 % ) , media comprises one additional cytokinin . In some embodi about 40 mg / L ( 10 % ), about 50 mg/ L ( 10 % ), about 60 ments , the media further comprises one or more auxin , such mg / L ( + 10 % ) , about 70 mg / L ( + 10 % ) , about 80 mg / L as NAA , 2,4 - D , IBA , IAA , picloram , or analogs thereof. In ( + 10 % ) , about 90 mg / L ( + 10 % ) , or about 100 mg / L ( + 10 % ) . other embodiments , the media useful for producing peren For example, the concentration of TDZ is about 0.2 ( -10 % ) nials , grasses and phyto - pharmaceutical plants does not to about 20 ( -10 % ) mg / L , about 0.4 ( 10 % ) to about 10 comprise a cytokinin or auxin . In yet other embodiments , the ( -10 % ) mg / L , or about 0.5 ( -10 % ) to about 2 ( -10 % ) mg / L . media useful for producing perennials, grasses and phyto [ 0402 ] In some embodiments, the concentration of TDZ or pharmaceutical plants does not comprise a plant hormone . analog thereof in the media useful for producing virus - free [ 0397 ] In some embodiments, the concentration of the plants, such as agricultural plants, is about 0.25 mg / L to strong cytokinin ( e.g. , TDZ ) in the media useful for produc about 100 mg / L , for example, about 0.5 mg / L to about 2 ing perennials, grasses and phyto - pharmaceutical plants , is mg / L . Thus, the concentration of TDZ or analog thereof can , about 0.25 mg / L ( + 10 % ) to about 100 mg / L ( + 10 % ) , for for example, be about 0.25 mg/ L , about 0.3 mg / L , about 0.4 example , is about 0.2 mg / L ( + 10 % ) , about 0.5 mg / L ( + 10 % ) , mg / L , about 0.5 mg / L , about 0.6 mg / L , about 0.7 mg/ L , about 1.0 mg / L ( + 10 % ) , about 5 mg / L ( + 10 % ) , about 10 about 0.8 mg / L , about 0.9 mg / L , about 1.0 mg / L , about out mg / L ( + 10 % ) , about 20 mg / L ( + 10 % ) , about 30 mg / L 1.5 mg / L , about 2.0 mg / L , about 2.5 mg / L , about 3 mg / L , ( -10 % ) , about 40 mg / L ( + 10 % ) , about 50 mg / L ( + 10 % ) , about 4 mg / L , about 5 mg / L , about 6 mg / L , about 7 mg / L , about 60 mg / L ( + 10 % ) , about 70 mg / L ( 10 % ) , about 80 about 8 mg / L , about 9 mg / L , about 10 mg / L , about 15 mg/ L , mg / L ( + 10 % ) , about 90 mg / L ( + 10 % ) , or about 100 mg / L about 20 mg / L , about 25 mg / L , about 30 mg / L , about 40 ( -10 % ) . For example , the concentration of TDZ is about 0.2 mg / L , about 50 mg / L , about 60 mg / L , about 70 mg / L , about ( -10 % ) to about 20 ( -10 % ) mg / L , about 0.4 ( -10 % ) to 80 mg / L , about 90 mg / L or about 100 mg / L . about 10 ( -10 % ) mg / L , or about 0.5 ( + 10 % ) to about 2 [ 0403 ] In some embodiments, the concentration of TDZ or ( + 10 % ) mg / L. analog thereof in the bud induction medium ( B001 ) is [ 0398 ] In some embodiments, the concentration of TDZ or effective to induce shoot buds . In some embodiments , the analog thereof in the media useful for producing perennials, concentration of TDZ or analog in the bud induction media US 2020/0383331 A1 Dec. 10 , 2020 27
( B001 ) thereof is about 0.25 mg / L to about 100 mg / L , for mg / L , about 0.25 mg/ L , about 0.3 mg / L , about 0.35 mg / L , example , about 0.5 mg / L to about 2 mg / L . Thus , the con about 0.4 mg / L , about 0.45 mg / L , about 0.5 mg / L , about 0.6 centration of TDZ or analog thereof in the bud induction mg / L , about 0.7 mg / L , about 0.8 mg / L , about 0.9 mg/ L , media ( B001 ) can , for example, be about 0.25 mg / L , about about 1.0 mg / L , about 1.5 mg / L , about 2.0 mg / L , about 2.5 0.3 mg / L , about 0.4 mg / L , about 0.5 mg/ L , about 0.6 mg / L , mg / L , about 3 mg / L , about 4 mg / L , about 5 mg / L , about 6 about 0.7 mg / L , about 0.8 mg / L , about 0.9 mg / L , about 1.0 mg / L , about 7 mg / L , about 8 mg / L , about 9 mg / L , about 10 mg / L , about out 1.5 mg / L , about 2.0 mg / L , about 2.5 mg / L , mg / L , about 15 mg/ L , about 20 mg / L , about 25 mg / L , about about 3 mg / L , about 4 mg / L , about 5 mg / L , about 6 mg / L , 30 mg / L , about 40 mg / L , about 50 mg / L , about 60 mg / L , about 7 mg / L , about 8 mg / L , about 9 mg / L , about 10 mg / L , about 70 mg/ L , about 80 mg / L , about 90 mg / L or about 100 about 15 mg / L , about 20 mg / L , about 25 mg / L , about 30 mg / L. mg / L , about 40 mg/ L , about 50 mg / L , about 60 mg / L , about [ 0409 ] In some embodiments, the concentration of the 70 mg / L , about 80 mg / L , about 90 mg / L or about 100 mg / L . auxin in the bud induction media ( B001 ) is about 0.01 [ 0404 ] In some embodiments, the bud induction medium mg / L ( + 10 % ) to about 50 mg / L ( + 10 % ) , for example, is ( B001 ) and / or the shoot elongation /maintenance medium about 0.01 mg / L ( + 10 % ) , about 0.05 mg / L ( + 10 % ) , about ( B002 ) further comprise one or more auxins. In some 0.1 mg/ L ( -10 % ) , about 0.5 mg / L ( + 10 % ) , about 1 mg / L embodiments , the auxins are selected from the group con ( + 10 % ) , about 5 mg / L ( + 10 % ) , about 10 mg / L ( + 10 % ) , sisting of B -naphthoxyacetic acid ( NAA ), 2,4 -Dichlorophe about 20 mg / L ( + 10 % ) , about 30 mg / L ( + 10 % ) , about 40 noxy acetic acid ( 2,4 - D ) , indole - 3 - butyric acid ( IBA ) , mg / L ( + 10 % ) , or about 50 mg / L ( -10 % ) . In some embodi indole - 3 - acetic acid ( IAA ), picloram , and analogs of each ments , the concentration of the auxin in the shoot elonga thereof. For example, the auxin is NAA or analogs thereof. tion /maintenance media ( BOO2 ) is about 0.01 ( -10 % ) to ( 0405 ] In some embodiments, the concentration of the about 20 ( -10 % ) mg / L , about 0.02 ( -10 % ) to about 10 auxin in the bud induction media ( B001 ) is about 0.01 ( + 10 % ) mg / L , or about 0.05 ( + 10 % ) to about 5 ( -10 % ) mg / L ( + 10 % ) to about 10 mg / L ( + 10 % ), for example , is mg / L . about 0.01 mg/ L ( + 10 % ) , about 0.05 mg / L ( + 10 % ) , about [ 0410 ] Non - limiting concentrations of the components in 0.1 mg/ L ( 10 % ) , about 0.5 mg/ L ( -10 % ) , about 1 mg / L the bud induction media ( B001 ) and shoot elongation / ( + 10 % ) , about 5 mg / L ( + 10 % ) , or about 10 mg / L ( + 10 % ) . maintenance media ( BOO2 ) are shown in Table 2. One or [ 0406 ] In some embodiments, the shoot elongation /main more components in table 2 can be omitted or replaced tenance media ( B002 ) comprises one or more cytokinins without affecting the function of the media . The concentra other than TDZ , such as meta - topolin , kinetin , isopente tion of each component can be adjusted without affecting the nyladenine ( ip , e.g. , 2ip ) , zeatin , trans -zeatin , zeatin ribo function of the media . side , dihydrozeatin , benzyleadenin ( BAP ) , benzyladenosine ( [ 9R ] BAP ) , analogs thereof. In some embodiments, the TABLE 2 shoot elongation /maintenance media ( BOO2 ) further com prise one or more auxin , such as NAA , 2,4 - D , IBA , IAA , Exemplary concentrations of bud induction media ( B001 ) picloram , or analogs thereof. and shoot elongation and maintenance media (BOO2 ) . [ 0407 ] In some embodiments, the concentration of cyto Shoot elongation kinin in the shoot elongation /maintenance media ( BOO2 ) is Bud Induction media & maintenance about 0.01 mg / L ( + 10 % ) to about 100 mg / L ( + 10 % ) , for Media ( B001 ) ( mg / L ) media ( B002 ) ( mg / L ) example , is about 0.01 mg / L ( + 10 % ) , about 0.05 mg / L NH4NO3 about 800 - about 2500 , about 800 - about 2500 , ( + 10 % ) , about 0.1 mg / L ( + 10 % ) , about 0.5 mg/ L ( + 10 % ) , e.g. , 1650 e.g. , 1650 KNO3 about 900 - about 3000 , about 900 - about 3000 , about 1 mg / L ( + 10 % ) , about 5 mg / L ( + 10 % ) , about 10 mg / L e.g. , 1900 e.g. , 1900 ( + 10 % ) , about 20 mg / L ( + 10 % ) , about 30 mg / L ( + 10 % ) , Ca ( NO3 ) 2 0 - about 800 , 0 -about 800, about 40 mg/ L ( + 10 % ) , about 50 mg / L ( -10 % ) , about 60 e.g. , 0 e.g. , O mg / L ( + 10 % ) , about 70 mg / L ( + 10 % ) , about 80 mg / L K2SO4 0 - about 800 , 0 -about 800 , e.g. , 0 e.g. , O ( + 10 % ) , about 90 mg / L ( + 10 % ) , or about 100 mg / L ( + 10 % ) . MgSO4 about 150 - about 550 , about 150 - about 550 , In some embodiments , the concentration of the cytokinin in e.g. , 370 e.g. , 370 the shoot elongation /maintenance media ( BOO2 ) is about MnSO4 about 8.0 - about 26.0 , about 8.0 - about 26.0 , 0.01 ( -10 % ) to about 20 ( -10 % ) mg / L , about 0.1 ( -10 % ) to e.g. , 16.9 e.g. , 16.9 ZnSO4 about 4.0 - about 12.0 , about 4.0 - about 12.0 , about 10 ( -10 % ) mg / L , or about 0.25 ( + 10 % ) to about 5 e.g., 8.6 e.g. , 8.6 ( + 10 % ) mg / L. CuSO4 about 0.010 - about 0.4 , about 0.010 - about 0.4 , [ 0408 ] In some embodiments, the concentration of the one e.g. , 0.025 e.g. , 0.025 CaCl2 about 200 - about 660 , about 200 - about 660 , or more cytokinins other than TDZ or an analog thereof in e.g. , 440 e.g. , 440 the shoot elongation /maintenance medium ( B002 ) is effec KI about 0.4 - about 1.5 , about 0.4 - about 1.5 , tive to elongate shoots. In some embodiments, the concen e.g. , 0.83 e.g. , 0.83 tration of the one or more cytokinins other than TDZ or an CoCl2 about 0.010 - about 0.4 , about 0.010 - about 0.4 , e.g. , 0.025 e.g. , 0.025 analog thereof in the shoot elongation /maintenance medium HZBO3 about 3.0 - about 9.0 , about 3.0 - about 9.0 , ( BOO2 ) from about 0.01 mg / L to about 100 mg / L , for e.g. , 6.2 e.g. , 6.2 example, from about 0.25 mg / L to about 5 mg/ L . Thus, the Na M004 about 0.10 - about 0.4 , about 0.10 - about 0.4 , concentration of one or more cytokinins other than TDZ or e.g. , 0.25 e.g. , 0.25 KH2PO4 about 80 - about 250 , about 80 - about 250 , analog thereof in the shoot elongation /maintenance media e.g. , 170 e.g. , 170 ( BOO2 ) can , for example, be about 0.01 mg/ L , about 0.02 K2SOS as necessary as necessary mg / L , about 0.03 mg / L , about 0.04 mg/ L , about 0.05 mg / L , FeSO4 about 25 - about 90 , about 25 - about 90 , about 0.06 mg/ L , about 0.07 mg / L , about 0.08 mg / L , about e.g. , 55.7 e.g. , 55.7 0.09 mg / L , about 0.10 mg / L , about 0.15 mg / L , about 0.20 US 2020/0383331 A1 Dec. 10 , 2020 28
TABLE 2 - continued is used more than one time . For example , certain embodi ments disclosed herein include cycling explants or shoots in Exemplary concentrations of bud induction media ( B001 ) a rotation of media . For example, an explant may be placed and shoot elongation and maintenance media (BOO2 ) . in a Stage 1 media followed by a Stage 2 media and then Shoot elongation returned back to its Stage 1 media . In this context, when Bud Induction media & maintenance exposure to a media is repeated , it retains its lowest Stage Media ( BOO1 ) ( mg / L ) media ( BO02 ) (mg / L ) number within the particular process . In some embodiments , Na EDTA about 35 - about 120 , about 35 - about 120 , the alternative media are selected from Stage 1 media , Stage e.g. , 74.6 e.g. , 74.6 Na H2PO4 about 80-250 , about 80-250 , 2 media , Stage 3 media , Stage 4 media , Stage 5 media , Stage e.g. , 170 e.g. , 170 6 media , Stage 7 media , etc. as described herein . myo - Inositol about 50 - about 150 , about 50 - about 150 , e.g. , 100 e.g. , 100 [ 0415 ] In some embodiments, the alternative media com Thiamine about 0.2 - about 0.6 , about 0.2 - about 0.6 , prise meta - topolin or an analogue thereof. In some embodi e.g, 0.4 e.g , 0.4 Sugar about 15 gr- about about 15 gr- about ments , the alternative media comprise at least two other 45 gr , e.g. 30 gr . 45 gr, e.g. 30 gr. cytokinins. In some embodiments, the alternative media agar (solid ) about 2.5 gr -about about 2.5 gr- about comprise at least three cytokinins. In some embodiments , 8.0 gr, e.g. , 4.5 gr 8.0 gr, e.g. , 4.5 gr said alternative media comprise at least one auxin and at PH about 5.5-6.5 , about 5.5-6.5 , least two cytokinins. In some embodiments, said alternative e.g., 5.7 e.g. , 5.7 media comprise at least two auxins and at least two cyto HORMONES kinins . In some embodiments , said alternative media com NAA about 0.05 about 0.01-50 prise at least two auxins and at least three cytokinins. In BAP about 1 about 0.01-50 TDZ about 0.25 - about 100 0 some embodiments , the media supports multiplication ST - 10 about 0.01 - about 50 about 0.01 - about 50 cycles for a predetermined period of time . In some embodi 2ip 0 about 6 ments, the media support multiplication cycles for at least six months. [ 0411 ] In some embodiments , the media can be selected [ 0416 ] In some embodiments, to begin the process , a Stage from the ones listed in the table shown in FIG . 13 , or any 1 media can be obtained or prepared. Stage 1 media include medium equivalent thereof. a pH that is generally hospitable to plants ( typically from [ 0412 ] In some embodiments , the bud induction media 4.0-7.0 or 4.5-6.5 ) . Stage 1 media disclosed herein can ( B001 ) comprise thidiazuron ( TDZ ) or analog thereof, and include ( i ) meta - topolin ; ( 2 ) at least three cytokinins ; ( 3 ) the the elongation and maintenance media comprise one or more cytokinin meta - topolin or an analogue thereof in combina cytokinins other than TDZ or an analog thereof. In some tion with at least two other cytokinins; ( 4 ) at least one auxin embodiments , the cytokinins other than TDZ are selected and at least two cytokinins; ( 5 ) at least two auxins and at from the group consisting of N6 -benzylaminopurine ( BAP ) , least two cytokinins or ( 6 ) at least two auxins and at least meta - topolin (mT ), zeatin , zeatin riboside, dihydrozeatin , three cytokinins. In certain embodiments, Stage 1 media kinetin , isopentenyladenine ( ip , e.g. , 2ip ) , adenine hemi must include more than 1 auxin . In other embodiments , sulfate, dimethylallyladenine, N-( 2 -chloro -4 -pyridyl )-N' Stage 1 media must include more than 1 cytokinin . In further phenylurea ) ( 4 - CPPU ) , and analogs thereof. In some embodiments, Stage 1 media must include more than 1 auxin embodiments , the media can be used for plants in vitro and more than 1 cytokinin . micropropagation of monocots or dicots . In some embodi [ 0417 ] In some embodiments, the cytokinins and auxins ments , the media can be used for bamboo plants in vitro are chosen from one or more of meta - topolin , thidiazuron , micropropagation . NAA , IBA , BAP , Zip , CCPU and DPU . In additional [ 0413 ] In some embodiments, the bud induction medium embodiments, the cytokinins and auxins are chosen from ( BOO1 ) comprises an effective amount of thidiazuron one or more of meta - topolin or analogues thereof, thidiaz ( TDZ ) or analog thereof, and wherein the shoot elongation / uron or analogues thereof, NAA or analogues thereof, IBA maintenance medium ( BOO2 ) comprises an effective or analogues thereof, BAP or analogues thereof, Zip or amount of one or more cytokinins other than TDZ or an analogues thereof, CCPU or analogues thereof and DPU or analog thereof. In some embodiments, the one or more analogues thereof. cytokinins other than TDZ or an analog thereof in the shoot [ 0418 ] In some embodiments, the media and at least two elongation /maintenance medium ( BOO2 ) is selected from other cytokinins. In some embodiments, the media supports the group consisting of N6 - benzylaminopurine ( BAP ) , multiplication cycles for at least six months. meta - topolin (mT ) , zeatin , zeatin riboside, dihydrozeatin , kinetin , 2 - isopentenyladenine ( 2ip ) , adenine hemi sulfate , [ 0419 ] In some embodiments, the media comprise at least dimethylallyladenine, N-( 2 - chloro - 4 -pyridyl ) -N ' -pheny three cytokinins . In some embodiments, the media can lurea ) ( 4 - CPPU ) , and analogs thereof. support multiplication cycles for at least six months . In some [ 0414 ] In addition to the “ bud induction media (B001 ) " embodiments, provided are media comprising the cytokinin and " shoot elongation /maintenance media ( B002) ” combi meta - topolin or an analogue thereof in combination with at nation described above , the present invention provides other least two other cytokinins. alternative media combinations for plant propagation . For [ 0420 ] In some embodiments, the media comprise at least example, provided are media combinations comprising at one auxin and at least two cytokinins . In some embodiments, least one Stage 1 medium and at least one Stage 2 medium . the media can support multiplication cycles for at least six In some embodiments, the number assigned to a media months. In some embodiments , at least one cytokinin is within a given process is maintained when a certain media meta - topolin or an analogue thereof. US 2020/0383331 A1 Dec. 10 , 2020 29
[ 0421 ] In some embodiments, the media comprise at least Chu's N - 6 , DKW , Gamborg's B - 5 , Hoaglands No. 2 , Kao & two auxins and at least two cytokinins. In some embodi Michayluk , Nitsch & Nitsch , Schenk and Hildebrant, Vacin ments, the media can support multiplication cycles for at and Went, Whites and WPM , available from commercial least six months . sources such as Caisson Laboratories, Inc or Phytotechnol [ 0422 ] In some embodiments, the media comprise at least ogy Laboratories. two auxins and at least three cytokinins. In some embodi [ 0428 ] One example of a Stage 1 media includes meta ments, the media can support multiplication cycles for at topolin . Another non - limiting example includes meta - topo least six months. lin , thidiazuron , NAA and BAP . Another non - limiting [ 0423 ] In some embodiments, the micropropagated plants example includes meta - topolin , NAA and BAP. Another are grown in vitro in sterile media . non - limiting example includes meta - topolin , NAA , BAP [ 0424 ] In some embodiments, the media can be liquid , and IBA . Another non - limiting example includes meta semi- solid , or solid , and the physical state of the media can topolin , thidiazuron , NAA , BAP and IBA . Another non be varied by the incorporation or removal of one or more limiting example includes thidiazuron , NAA , BAP and 2ip . gelling agents . Any gelling agent known in the art that is Another non - limiting example includes thidiazuron , NAA suitable for use in plant tissue culture media can be used . and 2ip . Another non - limiting example includes meta - topo Agar is most commonly used for this purpose . Examples of lin , thidiazuron , NAA , BAP, IBA and 2ip . Another non such agars include Agar Type A , E or M and Bacto® Agar limiting example includes meta - topolin , IBA , 2ip and BAP. ( Becton Dickinson & Co. ) . Other exemplary gelling agents Another non -limiting example includes meta - topolin , thidi include carrageenan , gellan gum ( commercially available as azuron , CPPU , NAA and BAP . Another non - limiting PhytaGelTM ( Sigma - Aldrich ), Gelriter ( Sigma - Aldrich ) and example includes meta - topolin , thidiazuron , DPU , NAA and GelzanTM ( Caisson Labs ) ) , alginic acid and its salts , and BAP . Another non - limiting example includes thidiazuron, agarose . Blends of these agents, such as two or more of agar, CPPU , BAP, IBA and 2ip . Another non - limiting example carrageenan , gellan gum , agarose and alginic acid or a salt includes CPPU , DPU , NAA and BAP . Another non - limiting thereof also can be used . example includes meta - topolin , thidiazuron , CPPU , DPU , [ 0425 ] Examples of plant growth regulators include NAA , BAP, IBA and 2ip . Each of these non - limiting abscisic acid ( ABA ) , triacontanol, phloroglucinol, auxins examples can also include analogues of meta - topolin , thidi and compounds with auxin - like activity, cytokinins and azuron , NAA , BAP, DPU , CPPU , IBA and /or 2ip . compounds with cytokinin - like activity . Exemplary auxins [ 0429 ] The Stage 1 media is then placed into test tubes or include 4 - fluorophenoxyacetic acid ( FA ), 2,4,5 - trichloro other appropriate containers (including jars, boxes , jugs , phenoxyacetic acid ( 2,4,5 - T ) , 3 - bromooxindole - 3 - aceito cups, sterile bag technology , bioreactors , temporary immer acid , 4 - bromophenoxyacetic acid , dicamba , p - chlorophe sion vessels , etc. wherein when not specified are collectively noxyacetic acid (CPA ) indole - 3 -propinoic acid ( IPA ), 2,4 referred to as “ tubes ” ). These tubes can be capped or covered dichlorophenoxyacetic acid ( 2,4 - D ) , indole - 3 - butyric acid and autoclaved to sterilize the tubes and media . In another ( IBA ) , indole - 3 - acetic acid ( IAA ), picloram and combina embodiment, sterilization is achieved by autoclaving at 5-25 tions thereof. Exemplary cytokinins include meta - topolin , pounds pressure psi at a temperature of 200 ° F. - for 200 ° F. thidiazuron, N- ( 2 - chloro - 4 -pyridyl ) -N - phenylurea ( CCPU ) , 10-25 minutes . In another embodiment, sterilization is 1,3 - diphenylurea ( DPU ) , adenine hemi sulfate, benzylad achieved by autoclaving at 15 pounds pressure psi at a enine, dimethylallyladenine , kinetin , zeatin , riboside , temperature of 250 ° F. for 15-18 minutes . Liquid media can adenosine, meta - topolin riboside , meta - topolin - 9 - glucoside , be subjected to filter sterilization . ortho - topolin , ortho - topolin riboside, ortho -topolin - 9 - gluco [ 0430 ] After the explants are allowed to establish them side , para - topolin , para -topolin riboside, para -topolin - 9 - glu selves on Stage 1 media , the cell cultures grown from the coside , ortho -methoxytopolin , ortho -methoxytopolin ribo explants are transferred into a Stage 2 media . Stage 2 media side, meta -methoxytopolin , meta -methoxytopolin riboside , disclosed herein can include ( i ) meta - topolin ; ( 2 ) at least meta -methoxytopolin - 9 - glucoside and combinations thereof three cytokinins ; ( 3 ) the cytokinin meta - topolin or an ana as well as plant extracts having cytokinin - like activity, such logue thereof in combination with at least two other cyto as coconut water, banana powder, malt extract, pineapple kinins ; ( 4 ) at least one auxin and at least two cytokinins ; ( 5 ) powder or tomato powder. at least two auxins and at least two cytokinins or ( 6 ) at least [ 0426 ] Gibberellic acid also can be included in the media . two auxins and at least three cytokinins . In certain embodi A sugar or combination of sugars can be included in the ments , Stage 2 media must include more than 1 auxin . In media and can serve as a carbon source . Such sugars are other embodiments, Stage 2 media must include more than known to those of ordinary skill in the art . Exemplary sugars 1 cytokinin . In further embodiments, Stage 2 media must include fructose , sucrose , glucose , maltose , galactose man include more than 1 auxin and more than 1 cytokinin . nitol and sorbitol or combinations thereof. Other exemplary [ 0431 ] In some embodiments, the cytokinins and auxins additives ( with suggested but non - limiting functions) are chosen from one or more of meta - topolin , thidiazuron , include polyamines ( regeneration enhancer ); citric acid , NAA , IBA , BAP, Zip , CCPU and DPU . In additional polyvinylpyrodine ( PVP ) and sodium thiosulfate ( anti embodiments , the cytokinins and auxins are chosen from browning agents ); CaNO3 or calcium gluconate (hyperhy one or more of meta - topolin or analogues thereof, thidiaz dricity reducer ); paclobutrazol or ancymidol (multiplication uron or analogues thereof, NAA or analogues thereof, IBA enhancer ); acetyl salicylic acid ( ethylene inhibitor) and or analogues thereof, BAP or analogues thereof, 2ip or p -chlorophenoxyisobutyric acid ( PCIB ) and triiodobenzoic analogues thereof, CCPU or analogues thereof and DPU or acid ( TIBA ) ( anti- auxins ). analogues thereof. [ 0427 ] In some embodiments , basal media can be [ 0432 ] In some embodiments, one example of a Stage 2 Murashige and Skoog ( MS ) . Suitable nutrient salts also media includes meta - topolin . Another non - limiting example include , without limitation , Anderson's Rhododendron, includes meta - topolin , thidiazuron , NAA and BAP. Another US 2020/0383331 A1 Dec. 10 , 2020 30 non - limiting example includes meta - topolin , NAA and BAP. embodiment, both Stage 1 and Stage 2 media include Another non - limiting example includes meta - topolin , NAA , meta - topolin , thidiazuron, NAA , BAP, IBA and 2ip . In BAP and IBA . Another non - limiting example includes meta another non - limiting embodiment, both Stage 1 and Stage 2 topolin , thidiazuron, NAA , BAP and IBA . Another non media include meta - topolin , IBA , 2ip and BAP . In another limiting example includes thidiazuron , NAA , BAP and 2ip . non -limiting embodiment, both Stage 1 and Stage 2 media Another non - limiting example includes thidiazuron , NAA include meta - topolin , thidiazuron , CPPU , NAA and BAP . In and 2ip . Another non - limiting example includes meta - topo another non -limiting embodiment, both Stage 1 and Stage 2 lin , thidiazuron , NAA , BAP, IBA and 2ip . Another non media include meta - topolin , thidiazuron , DPU , NAA and limiting example includes meta - topolin , IBA , 2ip and BAP . BAP . In another non - limiting embodiment, both Stage 1 and Another non - limiting example includes meta - topolin , thidi Stage 2 media include thidiazuron , CPPU , BAP, IBA and azuron , CPPU , NAA and BAP . Another non - limiting 2ip . In another non - limiting embodiment, both Stage 1 and example includes meta - topolin , thidiazuron , DPU , NAA and Stage 2 media include CPPU , DPU , NAA and BAP . In BAP . Another non - limiting example includes thidiazuron , another non - limiting embodiment, both Stage 1 and Stage 2 CPPU , BAP, IBA and 2ip. Another non - limiting example media include meta - topolin , thidiazuron , CPPU , DPU , includes CPPU , DPU , NAA and BAP . Another non - limiting NAA , BAP, IBA and 2ip . Each of these non - limiting example includes meta - topolin , thidiazuron , CPPU , DPU , examples can also include analogues of meta - topolin , thidi NAA , BAP , IBA and 2ip. Each of these non -limiting azuron , NAA , BAP , DPU , CPPU , IBA and / or 2ip . examples can also include analogues of meta - topolin , thidi [ 0434 ] In some embodiments , examples of Stage 1 and azuron , NAA , BAP, DPU , CPPU , IBA and / or 2ip . Stage 2 media are described in WO / 2011 / 100762 , which is [ 0433 ] In particular embodiments disclosed herein , both incorporated herein by references in its entirety. For Stage 1 and Stage 2 media include meta - topolin . In another example, Stage 1 and Stage 2 media can be selected from the non - limiting embodiment, both Stage 1 and Stage 2 media group consisting of Media BOO38 (i - v ); Media B0040 include meta - topolin , thidiazuron , NAA and BAP . In another ( i - v ) ; Media B0041 ( i - v ) ; Media BO036 ( i - v ) ; Media non - limiting embodiment, both Stage 1 and Stage 2 media BOO42 ( i - v ) ; Media BOO39 ( i - v ) ; Media BOO43 ( i - v ) ; include meta - topolin , NAA and BAP . In another non - limit Media BO044 ( i - v ) ; Media BO037 ( i - v ) ; Media BO031 ing embodiment, both Stage 1 and Stage 2 media include ( i - v ) ; Media BO028 ( i - v ) ; Media BO029 ( i - v ) ; Media meta - topolin , NAA , BAP and IBA . In another non - limiting BOO30 ( i - v ). embodiment, both Stage 1 and Stage 2 media include [ 0435 ] In some embodiments, examples of Stage 1 and meta - topolin , thidiazuron, NAA , BAP and IBA . In another non - limiting embodiment, both Stage 1 and Stage 2 media Stage 2 media are selected from the ones described below : include thidiazuron, NAA , BAP and 2ip . In another non limiting embodiment, both Stage 1 and Stage 2 media Media BOO38 (i - v ): include thidiazuron , NAA and 2ip. In another non - limiting [ 0436 ]
Standard Standard Standard Standard Standard Component BOO38 - i BOO38 - ii BOO38 - iii BOO38 - iv BO038 - V NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 Ca (NO3 )2 225-775 410-690 495-605 550 550 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 + 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 CaCl2 220-660 330-350 400-480 440 440 = 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 = .02 CoCl2 0.01-0.37 0.020-0.030 0.022-0.028 0.025 0.025 .002 H2B03 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 +.02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na2HPO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 +.02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Thidiazuron 0.36-1.12 0.56-0.94 0.67 - .083 0.75 0.75 +.02 Meta - topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2
Media B0040 ( i -v ) :
Standard Standard Standard Standard Standard Component BO040 - i BOO40 - ii BOO40 - iii BOO40 - iv BOO40 - V NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 US 2020/0383331 A1 Dec. 10 , 2020 31
-continued
Standard Standard Standard Standard Standard Component BOO40-1 BOO40-11 BOO40 - iii BOO4O BOO40 - V Ca ( NO3 ) 2 225-775 410-690 495-605 550 550 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8.0-26.0 12.0-22.0 15.0-19.0 16.9 16.9 + 0.2 ZnSO4 4.0-12.0 6.0-10.0 8.0-9.0 8.6 8.6 + 0.2 CuSO4 0.012-0.37 0.02-0.03 0.02-0.028 0.025 0.025 = .002 CaCl2 220-660 330-350 400-480 440 440 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 + .02 CoCl2 0.01-0.37 0.02-0.03 0.02-0.028 0.025 0.025 .002 HZBO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22-28 0.25 0.25 + .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 GO - 6 55.7 55.7 + 0.2 Na EDTA 37-112 74.6 74.6 + 0.2 Na H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 9.II 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 = .02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Meta - topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 Zq3 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2
Media B0041 (i - v ) : [ 0437 ]
Standard Standard Standard Standard Standard Component BOO41 - i BOO41 - ii BOO41-111 BOO41 - iv BOO41 - v NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 Ca ( NO3 ) 2 225-775 410-690 495-605 550 550 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 IKI 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022 - .028 0.025 0.025 + .002 CaCl2 220-660 330-350 400-480 440 440 + 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 + .02 CoCl2 0.01-0.37 0.02-0.03 0.02-0.028 0.025 0.025 + .002 HZBO3 3-9 4-8 G. 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 GO - 6 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-8 74.6 74.6 + 0.2 Na H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 9.II 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 NAA 0.05-0.15 0.07-0.12 0.09-0.11 0.1 0.1 + 0.02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 IBA 0.1-0.3 0.15-0.25 0.17-0.22 0.2 0.2 + 0.1 Meta - topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 Qq 3g 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 0.2
Media BO036 ( i - v ) :
Standard Standard Standard Standard Standard Component BOO36 - i BOO36 - ii BOO36 - i1i BOOZOI BOO36 - V NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8.0-26.0 12.0-22.0 15.0-19.0 16.9 16.9 + 0.2 ZnSO4 4.0-12.0 6.0-10.0 8.0-9.0 8.6 8.6 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 CaCl , 220-660 330-350 400-480 440 440 + 2 KI 0.4-1.25 0.6-1.05 0.7-0.90 0.83 0.83 + .02 CoCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 US 2020/0383331 A1 Dec. 10 , 2020 32
-continued
Standard Standard Standard Standard Standard Component BOO36 - i BOO36 - ii BOO36 - i1i BOO36 - iv BOO36 - V HZBO3 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 = .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 + .02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Thidiazuron 0.12-0.36 0.18-0.31 .22-28 0.25 0.25 + .02 Meta - topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2
Media B0042 ( i- v ): [ 0438 ]
Standard Standard Standard Standard Standard Component BOO42 - i BOO42-11 BOO42 - ili BOO42 - iv BOO42 - v NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 + 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 = .002 CaCl2 220-660 330-350 400-480 440 440 + 2 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 +0 CoCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 = .002 H3B03 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22-28 0.25 0.25 + .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 NAA 0.05-0.15 0.07-0.12 0.09-0.11 0.1 0.1 + 0.02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 IBA 0.1-0.3 0.15-0.25 0.17-0.22 0.2 0.2 + 0.1 Thidiazuron 0.12-0.36 0.18-0.31 .22-28 0.25 0.25 .02 Meta - topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9.6.1 5.5 5.5 + 0.2
Media BO039 ( i - v ): [ 0439 ]
Standard Standard Standard Standard Standard Component BOO39-1 BOO39 - ii BOO39 - iii BOO39 - iv BOO39 - V NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 – 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 CaCl2 220-660 330-350 400-480 440 440 + 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 = .02 CoCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 = .002 HZBO3 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 = .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 US 2020/0383331 A1 Dec. 10 , 2020 33
-continued
Standard Standard Standard Standard Standard Component BOO39-1 BOO39 - ii BOO39 - iii BOO39 - iv BOO39 - v Na H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 + .02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Meta - topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2 Silicon 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Solution mL
Media BOO43 (i - v ) : [ 0440 ]
Standard Standard Standard Standard Standard Component BOO43-1 BOO43-11 BOO43 - ili BOO43 - iv BOO43 -v NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 + 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 CaCl2 220-660 330-350 400-480 440 440 = 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 = .02 CoCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 = .002 H3B03 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 +.02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na2H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 = 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 = 0.2 NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 + .02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 IBA 0.1-0.3 0.15-0.25 0.17-0.22 0.2 0.2 + 0.1 Meta - topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2 Silicon 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Solution mL
Media B0044 ( i - v ): [ 0441 ]
Standard Standard Standard Standard Standard Component BOO44 - i BOO44 - ii BOO44 - ili BOO44 - iv BOO44 - v NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 + 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 CaCl2 220-660 330-350 400-480 440 440 + 2 KI 0.4-1.2 0.6-1.0 0.75-0.90 0.83 0.83 + .02 CoCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 HZBO3 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22-28 0.25 0.25 + .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 K2SOS 181.8-545.6 272.8-454.6 327.4-400.0 363.75 363.75 +.02 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 US 2020/0383331 A1 Dec. 10 , 2020 34
-continued
Standard Standard Standard Standard Standard Component BOO44 - i BOO44 - ii BOO44 - iii BOO44 - iv BOO44 - v Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 NAA 0.05-0.15 0.07-0.12 0.09-0.11 0.1 0.1 + 0.02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 IBA 0.1-0.3 0.15-0.25 0.17-0.22 0.2 0.2 + 0.1 Meta - topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Thidiazuron 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 = .02 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2
Media BOO37 ( i -v ) :
Standard Standard Standard Standard Standard Component BOO37 - i BOO37 - ii BOO37-112 BOO37 - iv BOO37 - v NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4.0-12.0 6.0-10.0 8.0-9.0 8.6 8.6 + 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 CaCl2 220-660 330-350 400-480 440 440 + 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 +.02 CoCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 H3B03 3-9 4-8 5-7 6.2 6.2 + 0.2 NazM004 0.12-0.36 0.18-0.31 .22-28 0.25 0.25 + .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37. - 112 55-94 67-82 74.6 74.6 + 0.2 Na2H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 +.02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Meta - topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2
Media BO031 ( i - v ) :
Standard Standard Standard Standard Standard Component BOO31 - i BOO31-11 BOO31-111 BOO31 - iv BOO31 - V NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 + 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 = .002 CaCl2 220-660 330-350 400-480 440 440 + 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 +.02 CoCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 = .002 H2B03 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 + .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 + .02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Thidiazuron 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Meta- topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g/ L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2 US 2020/0383331 A1 Dec. 10 , 2020 35
Media BO028 ( i -v ) :
Standard Standard Standard Standard Standard Component BOO28 - i BOO28 - ii BOO28-111 BOO28 - iv BOO28 - V NH4NO3 600-1800 900-1500 1080-1320 1200 1200 + 2 Ca ( NO3 ) 2 838-2515 1257-2096 1510-1844 1677 1677 + 2 K2SO4 121-363 181-302 218-266 242 242 + 2 MgSO4 270-830 410-690 500-610 555 555 + 2 MnSO4 12.6-38.0 19.0-31.7 22.8-27.8 25.35 25.35 +.02 ZnSO4 6.4-19.5 9.6-16.2 11.5-14.0 12.9 12.9 + 0.2 CuSO4 0.01-0.05 0.02-0.04 0.033-0.041 0.037 0.037 + 0.002 CaCl2 48-144 72-120 85-105 96 96 + 2 HZBO3 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22-28 0.25 0.25 + .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27.0-84.0 40.0-70.0 50.0-60.0 55.7 55.7 + 0.2 Na EDTA 37.0-112.0 55.0-94.0 67.0-82.0 74.6 74.6 + 0.2 Na H2PO4 42-128 63-106 75-95 85 85 + 2 myo - Inositol 100-300 150-250 180-220 200 200 + 2 Thiamine 0.4-1.4 0.6-1.1 0.8-1.0 0.9 0.9 + 0.2 Pyridoxine 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Nicotinic acid 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Glycine 1-3 1.5-2.5 1.75-2.25 2 2 + 1 Riboflavin 10-30 15-25 18-22 20 20 2 BAP 0.1-0.3 0.15-0.25 0.17-0.22 0.2 0.2 + 0.1 NAA 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 0.2 Thidiazuron 0.36-1.12 0.56-0.94 0.67-083 0.75 0.75 + .02 2ip 7-23 11-19 13-17 15 15 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Carrageenan g / L 3-11 4-10 5-8 7 7 + 2
Media B0029 ( i- v ):
Standard Standard Standard Standard Standard Component BOO29-1 BOO29 - ii BOO29 - iii BOO29 - iv BOO29 - V NH4NO3 600-1800 900-1500 1080-1320 1200 1200 + 2 Ca ( NO3 ) 2 838-2515 1257-2096 1510-1844 1677 1677 + 2 K2SO4 121-363 181-302 218-266 242 242 + 2 MgSO4 270-830 410-690 500-610 555 555 + 2 MnSO4 12.6-38.0 19.0-31.7 22.8-27.8 25.35 25.35 = .02 ZnSO4 6.4-19.5 9.6-16.2 11.5-14.0 12.9 12.9 + 0.2 CuSO4 0.01-0.05 0.02-0.04 0.03-0.04 0.037 0.037 = .002 CaCl , 48-144 72-120 85-105 96 96 + 2 H2B03 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 = .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na H2PO4 42-128 63-106 75-95 85 85 + 2 myo - Inositol 100-300 150-250 180-220 200 200 + 2 Thiamine 0.4-1.4 0.6-1.1 0.8-1.0 0.9 0.9 + 0.2 Pyridoxine 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Nicotinic acid 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Glycine 1-3 1.5-2.5 1.75-2.25 2 2 + 1 Riboflavin 10-30 15-25 18-22 20 20 = 2 BAP 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 NAA 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Thidiazuron 0.36-1.12 0.56-0.94 0.67-083 0.75 0.75 +.02 2ip 10-30 15-25 18-22 20 20 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Carrageenan g / L 3-11 4-10 5-8 7 7 + 2
Media BO030 ( i -v ) :
Standard Standard Standard Standard Standard Component BOO30 - i BOO30 - ii BOO30 - iii BOO30 - iv BOO30 - V NH4NO3 600-1800 900-1500 1080-1320 1200 1200 + 2 Ca ( NO3 ) 2 838-2515 1257-2096 1510-1844 1677 1677 + 2 K2SO4 121-363 181-302 218-266 242 242 + 2 US 2020/0383331 A1 Dec. 10 , 2020 36
-continued
Standard Standard Standard Standard Standard Component BOO30 - i BOO30 - ii BOO30 - iii BOO30 - iv BOO30 - V MgSO4 270-830 410-690 500-610 555 555 + 2 MnSO4 12.6-38.0 19.0-31.7 22.8-27.8 25.35 25.35 + .02 ZnSO4 6.4-19.5 9.6-16.2 11.5-14.0 12.9 12.9 + 0.2 CuSO4 0.01-0.05 0.02-0.04 0.033-0.041 0.037 0.037 = .002 CaCl2 48-144 72-120 85-105 96 96 + 2 HZBO3 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 +.02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na H2PO4 42-128 63-106 75-95 85 85 + 2 myo - Inositol 100-300 150-250 180-220 200 200 + 2 Thiamine 0.4-1.4 0.6-1.1 0.8-1.0 0.9 0.9 + 0.2 Pyridoxine 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 0.2 Nicotinic acid 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Glycine 1-3 1.5-2.5 1.75-2.25 2 2 + 1 Riboflavin 10-30 15-25 18-22 20 20 + 2 NAA 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Thidiazuron 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 .02 2ip 2.5-7.5 3.7-6.2 4.5-5.5 naina5 5 2 Sugar g / L 12-37 15-35 20-30 25 25 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2 Carrageenan g / L 1-3 1.5-2.5 1.75-2.25 2 2 + 1
Media BO035 [ 0442 ]
Standard Standard Standard Standard Standard Component BOO35 - i BOO35 - ii BOO35-111 BOO35 - iv BOO35 - v NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 K2SO4 242-726 363-605 436-532 484 484 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 + 0.2 CuSO4 0.01-0.37 0.02-0.03 0.02-0.028 0.025 0.025 = .002 CaCl2 220-660 330-350 400-480 440 440 + 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 .02 CoCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 .002 HZBO3 3.0-9.0 4.0-8.0 5.0-7.0 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22-28 0.25 0.25 = .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 100-300 150-250 180-220 200 200 + 2 Thiamine 0.45-1.35 0.67-1.13 0.8-1.0 0.9 0.9 + 0.2 NAA 0.15-0.45 0.22-0.38 0.27-0.33 0.3 0.3 + 0.2 BAP 1-3 1.25-2.75 1.5-2.5 2 2 + 0.5 Thidiazuron 0.25-0.75 0.37-0.63 0.45-0.55 0.5 0.5 1.2 Meta - Topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2
Media BO038 CPPU [ 0443 ]
Standard Standard Standard Standard Standard BOO3 BOO38 BOO38 BOO38 BOO38 Component CPPU - i CPPU - ii CPPU - 111 CPPU - iv CPPU - V NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 Ca ( NO3 ) 2 225-775 410-690 495-605 550 550 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 US 2020/0383331 A1 Dec. 10 , 2020 37
-continued
Standard Standard Standard Standard Standard BOO3 BO038 BOO38 BOO38 BOO38 Component CPPU - i CPPU - ii CPPU - 111 CPPU - iv CPPU - V MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 = .002 CaCl2 220-660 330-350 400-480 440 440 + 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 = .02 CoCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 .002 HZBO3 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 +.02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na2H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 Casein 1-3 1.5-2.5 1.75-2.25 2 2 + 1 Hydroxylate g / L NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 + .02 BAP 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.2 Thidiazuron 0.36-1.12 0.56-0.94 0.67 - .083 0.75 0.75 +.02 Meta - Topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 CPPU 0.36-1.12 0.56-0.94 0.67 - .083 0.75 0.75 + .02 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2
Media BO038 DPU [ 0444 ]
Standard Standard Standard Standard Standard BO038 BOO38 BOO38 BOO38 BO038 Component CPU - I CPU - ii CPU - ili CPU - iv CPU - V NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 Ca ( NO3 ) 2 225-775 410-690 495-605 550 550 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 15-19 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8-9 8.6 8.6 + 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 = .002 CaCl2 220-660 330-350 400-480 440 440 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 +.02 COCl2 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 HZBO3 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 = .02 KH2PO4 85-255 120-210 150-190 170 170 + 2 FeSO4 27-84 40-70 50-60 55.7 55.7 + 0.2 Na EDTA 37-112 55-94 67-82 74.6 74.6 + 0.2 Na2H2PO4 85-255 120-210 150-190 170 170 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 + 0.2 Casein 1-3 1.5-2.5 1.75-2.25 2 2 + 1 Hydroxylate g / L NAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 + .02 BAP 0.5-1.5 0.7- 1.3 0.9-1.1 1 1 + 0.2 Thidiazuron 0.36-1.12 0.56-0.94 0.67-083 0.75 0.75 +.02 Meta - Topolin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 DPU 0.36-1.12 0.56-0.94 0.67-083 0.75 0.75 +.02 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 + 0.2
[ 0445 ] In some embodiments , the non - cytokinin compo and others will lack nitrates. In some embodiments , the nents are those found in Anderson's Rhododendron , Chu's media have higher or lower levels of macronutrients and N - 6 , DKW , Gamborg's B - 5 , Hoaglands No. 2 , Kao & lack nitrates. In some embodiments , the media have higher Michayluk , Nitsch & Nitsch , Schenk and Hildebrant, Vacin levels of macronutrients and lack nitrates . and Went, Whites and WPM , available from commercial [ 0446 ] Media disclosed herein also include spiked media . sources . Particular media can have higher or lower levels of Spiked media are those in which the concentration of at least macronutrients than those provided in the preceding tables one cytokinin and /or auxin in the media described above is US 2020/0383331 A1 Dec. 10 , 2020 38 increased by, for example and without limitation , 1 % , 3 % , -continued 5 % , 7 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % 95 % , 100 % , Spiked Spiked Spiked Spiked 105 % , 110 % or 200 % . In other embodiments , the concen Spiked tration of at least one cytokinin and / or auxin in the media Component BOO41 - i BOO41 - ii BOO41-111 BOO41 - iv BOO41 - V described above is increased by, for example and without limitation , 1-10 % , 5-15 % , 10-20 % , 15-25 % , 20-30 % , IBA 0.2 0.75 0.2 0.5 1 25-35 % , 30-40 % , 35-45 % , 40-50 % , 45-55 % , 50-60 % , Meta - topolin 8 16 9 30 5 55-65 % , 60-70 % , 65-75 % , 70-80 % , 75-85 % , 80-90 % , 85-95 % , 90-100 % , 95-105 % , 100-110 % , 105-115 % , 110 120 % , 115-125 % , 120-130 % , 125-135 % , 130-140 % , 135 145 % , 140-150 % , 145-155 % , 150-160 % , 155-165 % , 160 Media BO036 ( i - v ) : 170 % , 165-175 % , 170-180 % , 175-185 % , 180-190 % , 185 195 % , 190-200 % , 195-205 % , 3-6 % , 7-17 % , 12-22 % , 17-27 % , 22-32 % , 27-37 % , 32-42 % , 37-47 % , 42-52 % , Spiked Spiked Spiked Spiked Spiked 47-57 % , 52-62 % , 57-67 % , 62-72 % , 67-77 % , 72-82 % , Component BOO36 - i BOO36 - ii BO036 - iii BOO36 - iv BOO36 - V 77-87 % , 82-92 % , 87-97 % , 92-102 % , 97-107 % , 102-112 % , 107-117 % , 112-122 % , 117-127 % , 122-132 % , 127-137 % , NAA 1 2 0.5 0.25 0.05 132-142 % , 137-147 % , 142-152 % , 147-157 % , 152-162 % , 157-167 % , 162-172 % , 167-177 % , 172-182 % , 177-187 % , BAP 11 5 50 10 1 162-172 % , 167-177 % , 182-192 % , 187-197 % , 192-202 % , Thidiazuron 0.5 1 1.5 1.75 0.25 197-207 % , or 200-210 % . When more than one cytokinin Meta - topolin 9 8 7 6 500 and / or auxin is spiked , the concentrations of each raised cytokinin and / or auxin can be raised by the same amount or a different amount than other cytokinins and / or auxins in the media . Media BOO42 ( i - v ) : [ 0447 ] The following tables provide non -limiting examples of spiked media disclosed herein . Each media [0450 ] includes the components described in its respective table above or adjusted as described in the next paragraph with the following adjustments to cytokinin levels . Spiked Spiked Spiked Spiked Spiked Component BOO42 - i BOO42 - ii BOO42 - iii BOO42 - iv BOO42 - v Media BO038 (i - v ) : NAA 0.1 0.1 0.1 0.1 0.1 BAP 1 1 2 3 4 IBA 0.4 0.2 1 0.5 0.2 [ 0448 ] Thidiazuron 0.25 0.5 1 5 0.3 Meta - topolin 50 40 5 15 10 Spiked Spiked Spiked Spiked Spiked Component BOO38 - i BOO38 - ii BOO38 - iii BOO38 - iv BOO38 - V NAA 0.1 0.1 0.05 0.05 0.5 BAP 2 1 1.25 1 1.5 Media BOO39 (i - v ) : Thidiazuron 0.75 0.7 1 0.75 1.25 Meta - topolin 10 5 5 6 7.5 [ 0451 ]
Media B0040 ( i - v ): Spiked Spiked Spiked Spiked Spiked Component BOO39-1 BOO39 - ii BOO39-1ii BOO39 - iv BOO39 - V NAA 2 0.05 0.05 10 0.15 Spiked Spiked Spiked Spiked Spiked BAP 1 100 150 80 5 Component BOO40 - i BOO40 - ii BO040-11i BOO40 - iv BOO40 - V Meta - topolin 85 300 100 5 25 NAA 0.05 0.05 0.2 0.1 0.05 BAP 1 2 1 2 5 Meta - topolin 20 10 7.5 10 5 Media B0043 ( i - v ): Media BOO41 ( i - V ): [ 0452 ] [ 0449 ] Spiked Spiked Spiked Spiked Spiked Component BOO43 - i BOO43 - ii BOO43 - lii BOO43 - iv BOO43 - V Spiked Spiked Spiked Spiked Spiked NAA 0.05 0.15 0.05 0.1 Component BOO41-1 BOO41 - ii BOO41-11i BOO41 - iv BOO41 - V BAP 1 1 5 1 IBA 0.2 0.4 0.2 1 NAA 0.1 0.1 0.4 0.1 0.5 Meta - topolin 1000 890 75 30 mtinin BAP 1 1 3 1 2 US 2020/0383331 A1 Dec. 10 , 2020 39
Media BO044 ( i - v ) : Media BO030 ( i -v ) : [ 0453 ] Spiked Spiked Spiked Spiked Spiked Component BOO30 - i BO030 - ii BOO30 - iii BOO30 - iv BOO30 - V Spiked Spiked Spiked Spiked Spiked Component BOO44 - i BO044 - ii B0044-11i BO044 - iv BOO44 - v NAA 1 1 4 10 1 Thidiazuron 1 2 5 10 0.25 NAA 0.1 0.1 2 0.4 0.5 2ip 15 7.5 5 5 5 BAP 3 1 4 1.5 2 IBA 0.5 0.2 0.4 0.3 1 Thidiazuron 0.25 0.25 0.5 10 3 Meta - topolin 450 20 10 7.5 Media BO035 [ 0454 ]
Spiked Spiked Spiked Spiked Spiked Component BOO35-1 BOO35 - ii BOO35 - iii BOO35 - iv BOO35 - v NAA 1 0.6 0.3 0.4 0.30 BAP 4 3 1.5 2 1 Thidiazuron 0.75 0.25 0.5 0.5 0.25 Meta - Topolin 10 5 5 6 5
Media BOO37 ( i - v ): Media BO038 CPPU [ 0455 ] Spiked Spiked Spiked Spiked Spiked Component BOO37 - i BO037 - ii BO037 - iii BOO37 - iv BOO37 - v Spiked Spiked Spiked Spiked Spiked NAA 0.05 0.05 1 20.25 0.05 BO038 BOO38 BO038 BO038 BOO38 BAP 1.3 3 1.6 2 1 Component CPPU - i CPPU - ii CPPU - ili CPPU - iv CPPU - V Meta - topolin 65 70 75 80 20 NAA 0.75 1 0.05 1 0.05 BAP 1.25 2 1 1 2 Thidiazuron 1 1.5 1 0.75 1.5 Media BO031 ( i -v ) : Meta - Topolin 6 10 8 5 5 CPPU 0.8 1.5 1.5 0.75 0.75 Spiked Spiked Spiked Spiked Spiked Component BOO31 - i BOO31 - ii BOO31-111 BOO31 - iv BOO31 - v Media BOO38 DPU NAA 0.05 0.5 1 1.75 0.05 BAP 4 3 2 1 1 [ 0456 ] Thidiazuron 5 10 15 20 0.5 Meta - topolin 750 43 12 300 125 Spiked Spiked Spiked Spiked Spiked BOO38 BO038 BO038 BOO38 BO038 Media BOO28 ( i - v ) : Component CPU - I CPU - ii CPU - lii CPU - iv CPU - v NAA 0.1 0.1 1.8 3 0.3 BAP 1.5 2 1.8 2 1.5 Spiked Spiked Spiked Spiked Spiked Thidiazuron 1.5 1.5 1.8 17 3 Component BOO28-1 BOO28 - ii BOO28-11i BOO28 - iv BOO28 - v Meta - Topolin 10 9 5 6 5 BAP 0.2 0.4 0.6 0.8 0.3 DPU 4 Örnom3 1.8 0.75 0.75 NAA 3 0.75 0.5 1 0.5 Thidiazuron 7.5 75 9 150 3.5 30 [ 0457 ] Additional spiked media can include any standard 2ip 80 60 45 20 media described above with the addition or adjustment to the following cytokinin and / or auxin concentrations: Media BO029 ( i - v ): Media AA [ 0458 ] Spiked Spiked Spiked Spiked Spiked Component BOO29 - i BOO29 - ii BOO29 - iii BOO29 - iv BOO29 - V BAP 30 10 7 5 5 Component AA - i AA - ii ?? - iii NAA 4 3 2 1 1 Thidiazuron 0.75 75 7.5 5 0.75 BAP 1 1 1 2ip 60 20 80 Bunu40 25 Thidiazuron 2.5 5 10 US 2020/0383331 A1 Dec. 10 , 2020 40
Media AB [ 0462 ] One or more compositions of the media disclosed herein can also be adjusted based on the plant species . [ 0459 ] [ 0463 ] In some embodiments, the media disclosed herein can be used for bamboo tissue culture. Representative gen AB - ii AB - iii era of bamboo are described in WO / 2011 / 100762 , which is Component AB - i AB - iv incorporated herein by reference in its entirety . BAP 5 10 20 40 [ 0464 ] In some embodiments , Stage 1 media are selected from the group consisting of BO038 - v media , BO038 CPPU - v media , BOO38 DPU - V, BOO35 - v media or Media AC BOO37 - v media or spiked versions thereof. [ 0465 ] In some embodiments, Stage 2 media are selected [ 0460 ] from the group consisting of BOO39 - v media ; BO040 - V media ; BOO41 - v media ; BOO42 - v media ; BOO43 - v media ; BOO44 - v media , BOO38 - CPPU - v media , BOO38 DPU - V , Component AC - i AC - ii AC - iii AC - iv AC -V BO035 - v media or spiked and reduced / standard versions BAP 1 1 1 1 1 thereof for 10-120 day cycles ( as modified for spiked media CPPU 2.5 7.5 10 25 50 as described more fully below ) . [ 0466 ] In some embodiments, one or more Stage 3 media [ 0461 ] As explained more fully below , when a spiked can be included . In some embodiments , Stage 3 media are media is utilized , explants or shoots generally ( but not selected from the group consisting of B0046 - v media ; necessarily ) remain on the spiked media for a shorter period B0045 - v media or B0047 - v media or spiked and reduced / of time than those kept on non - spiked media and following standard versions thereof ( as modified for spiked media as culture on a spiked media , the explants or shoots are described more fully below ) . transferred to a media containing standard , reduced or no [ 0467 ] In some embodiments, non - limiting examples of levels of cytokinins and / or auxins ( those containing reduced Stage 2 , Stage 3 , Stage 4 , Stage 5 , Stage 6 , Stage 7 media or no cytokinins and / or auxins are both referred to as include: “ reduced ” media herein ) . Media BO045 ( i - v ) :
Standard Standard Standard Standard Standard Component BOO45 - i BOO45 - ii BOO45 - iii BOO45- iv BOO45 - v NH4NO3 825-2475 1237-2063 1485-1815 1650 1650 + 2 KNO3 950-2850 1425-2375 1710-2090 1900 1900 + 2 MgSO4 185-555 275-465 330-410 370 370 + 2 MnSO4 8-26 12-22 IG . 16.9 16.9 + 0.2 ZnSO4 4-12 6-10 8 . 8.6 8.6 + 0.2 CuSO4 0.01-0.37 0.02-0.03 0.022-0.028 0.025 0.025 + .002 CaCl2 220-660 330-350 400-480 440 440 + 2 KI 0.40-1.25 0.60-1.05 0.75-0.90 0.83 0.83 .02 CoCl2 0.012-0.378 0.020-0.030 0.022-0.028 0.025 0.025 + .002 H3B03 3-9 4-8 E. 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 QQQ 0.25 0.25 = .02 KH2PO4 85-255 120-210 150-190 170 170 = 2 FeSO4 13-42 20-34 253 27.8 27.8 + 0.2 Na EDTA 18-56 28-46 33-4 37.3 37.3 + 0.2 myo - Inositol 50-150 75-125 WO II 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.35-0.45 0.4 0.4 + 0.2 Pyridoxine 0.2-0.8 0.3-0.7 04-0 . 0.5 0.5 0.2 Nicotinic acid 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Glycine 1-3 1.5-2.5 1.75-2.25 2 2 + 1 NAA 0.05-0.15 0.07-0.12 0.09-0.11 0.1 0.1 + 0.02 IAA 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 .02 Sugar g / L 15-45 22-37 30 30 + 2 Agar g / L 2.7-8.2 4.1-6.8 4.9-6.1 5.5 5.5 = 0.2
Media B0046 ( i -v ) :
Standard Standard Standard Standard Standard Component BOO46 - i BOO46 - ii BOO46 - iii BOO46 IV BOO46 - V NH4NO3 700-2100 1050-1750 1260-1540 1400 1400 + 2 Ca ( NO3 ) 2 973-2919 1459-2433 1752-2140 194 1946 = 2 K2SO4 606-1818 909-1515 1091-1333 1212.5 1212.5 0.2 MgSO4 370-1110 555-925 665-815 740 740 + 2 MnSO4 16.9-50.7 25.4-42.3 30.5-37.1 33.8 33.8 + 0.2 ZnSO4 8.6-25.8 12.9-21.5 15.5-18.0 17.2 17.2 + 0.2 CuSO4 0.02-0.08 0.03-0.07 0.04-0.06 0.05 0.05 +.02 CaCl2 72-216 108-180 130-158 144 144 + 2 US 2020/0383331 A1 Dec. 10 , 2020 41
-continued Standard Standard Standard Standard Standard Component BOO46 - i BOO46 - ii BOO46-111 BOO46 - iv BOO46 - v HZBO3 3-9 4-8 5-7 6.2 6.2 + 0.2 Na M004 0.12-0.36 0.18-0.31 .22 - .28 0.25 0.25 +.02 KH2PO4 72-342 202-338 243-297 270 270 + 2 FeSO4 16.68-50.04 25.02-41.70 30.06-36.66 33.36 33.36 + .02 Na EDTA 22.38-67.14 33.57-55.95 40.36-49.16 44.76 44.76 + .02 myo - Inositol 100-300 150-250 180-220 200 200 + 2 Thiamine 0.4-1.4 0.6-1.2 0.8-1.0 0.9 0.9 + 0.2 Pyridoxine 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Nicotinic acid 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Glycine 1-3 1.5-2.5 1.75-2.25 2 2 + 1 Riboflavin 10-30 15-25 18-22 20 20 + 2 Ascorbic Acid 50-150 75-125 90-110 100 100 + 2 NAA 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Carrageenan g / L 4-12 6-10 7-9 8 8 2 Charcoal gL 150-450 220-370 270-330 300 300 2
Media BO047 ( i -v ) :
Standard Standard Standard Standard Standard Component BOO47 - i BOO47 - ii BOO47 - iii BOO47 - iv BOO47 - v NH4NO3 410-1240 620-1030 740-910 825 825 + 2 Ca ( NO3 ) 2 475-1425 710-1190 855-1045 950 950 + 2 MgSO4 90-280 140-230 160-200 185 185 + 2 MnSO4 4.20-12.70 6.30-10.60 7.65-9.25 8.45 8.45 +.02 ZnSO4 2.0-6.5 3.0-5.5 3.5-5.0 4.3 4.3 0.2 CuSO4 .0062-0188 .0094 - .0156 .0115 - .0135 0.0125 0.0125 + .0002 CaCl2 110-330 165-285 195-240 220 220 + 2 KI .20 - .62 .31 - .52 .37 - .45 0.415 0.415 + .002 H3B03 1.5-4.6 2.3-4.0 2.8-3.4 3.1 3.1 + 0.2 CaCl2 .006 - .018 .009 - .015 .011-013 0.0125 0.0125 + .0002 Na M004 .06 - .18 .09 - .15 .11-13 0.125 0.125 + .002 KH2PO4 40-130 60-110 75-95 85 85 + 2 FeSO4 6.8-20.9 10.4-17.5 12.5-15.5 13.9 13.9 + 0.2 Na EDTA 9.3-27.9 13.9-23.3 16.8-20.4 18.65 18.65 + .02 Na H2PO4 40-130 60-110 75-95 85 85 + 2 myo - Inositol 50-150 75-125 90-110 100 100 + 2 Thiamine 0.2-0.6 0.3-0.5 0.36-0.44 0.4 0.4 +.2 Pyridoxine 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 Nicotinic acid 1-3 1.5-2.5 1.75-2.25 2 2 + 1 Riboflavin 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 NAA 0.2-0.8 0.3-0.7 0.4-0.6 0.5 0.5 + 0.2 IBA 0.5-1.5 0.7-1.3 0.9-1.1 1 1 + 0.5 Sugar g / L 15-45 22-37 27-33 30 30 + 2 Agar g / L 1.5-4.5 2.0-4.0 2.5-3.5 3 3 + 2 Carrageenan g / L 2.5-7.5 3.7-6.2 4.5-5.5 5 5 + 2 Charcoal g / L 2.5-7.5 3.7-6.2 4.5-5.5 5 + 2
[ 0468 ] In some embodiments, non - limiting examples of Media BO046 ( i - v ) : spiked versions of these media include: Spiked Spiked Spiked Spiked Spiked Media B0045 ( i - v ) : Component BOO46 - i BOO46 - ii BOO46-11i BOO46 - iv BOO46 - V NAA 1 1.5 2 50 5 Spiked Spiked Spiked Spiked Spiked Component BOO45 - i BOO45 - ii BO045-111 BO045 - iv BOO45 - v Media B0047 ( i - v ) : Spiked Spiked Spiked Spiked Spiked NAA 0.2 0.1 0.1 0.3 2 Component BOO47 - i BOO47 - ii BOO47 - iii BO047 - iv BOO47 - v IAA 0.5 0.5 1 0.05 0.75 NAA 1 1.5 0.5 0.5 3 IBA 12 10 5 2 1 US 2020/0383331 A1 Dec. 10 , 2020 42
Cytokinins and Analogs aryl or heteroaryl each optionally substituted with a C1 - C6 [ 0469 ) Compounds useful according to the present disclo alkyl, SH , NHR3 , CO2R3 or halogen ; sure include meta - topolin analogues having a general for R3 is H , OH , C1 - C6 alkyl, C1 - C6 alkylene, C1 - C6 alkynyl, mula halogen , carboxylic group , ester group , aldehyde or cyano ; p is 0 to 5 ; and q is 0 to 6 . HN - R1 - Y [ 0476 ] In some embodiments , the compounds have a structure , W (R2 ) , (R4 ) , HN - (CH2 ), wherein W is an aryl or heteroaryl; 7 R1 is substituted or unsubstituted alkyl wherein any C in the alkyl can be substituted with 0 , N or S ; each R2 is independently H , OH , C1 - C6 alkyl, C1 - C6 X alkylene, C1 - C6 alkynyl, halogen , cyano , C1 - C6 alkyloxy, (R2 ) , aryl or heteroaryl each optionally substituted with a C1 - C6 alkyl, SH , NHR3 , CO2R3 or halogen ; R3 is H , OH , C1 - C6 alkyl, C1 - C6 alkylene, C1 - C6 alkynyl, [ 0477 ] In some embodiments, the compounds have a halogen , carboxylic group , ester group , aldehyde or cyano ; structure , r is 0 to 8 . [ 0470 ] In some embodiments, W is (R4 )p tat X HN— (CH2 ) ? o ats xat N 731 84 or X43 [ 0471 ] wherein a dashed line represents the presence or ( R2) absence of a bond ; [ 0472 ] X1 - X ' is each independently selected from C , N , O , S with the proviso that the X linking the ring to N is C. [ 0478 ] Further still , compounds can have structures [ 0473 ] In some embodiments, the compounds have a selected from structure , ( R4) p , HN — R - Y HN - ( CH2) ? 7 X2X ' : - ** X3 - (X R2 ) NH2 wherein a dashed line represents the presence or absence of (R4 ) p2 a bond . HN- (CH2 ) ? [ 0474 ] In some embodiments, the compounds have a } structure , N X12 – X11 ( R4) p HN— (CH2 ) a tro ( R4 ), x' : - ** ( -X9 HN- (CH2 ) ? It's o X3 — X4 (R2 ) , N [ 0475 ] wherein a dashed line represents the presence or NH absence of a bond ; X8 - X12 is each independently selected from C , N , O , S ; H or each R4 is independently H , OH , C1 - C6 alkyl, C1 - C6 alkylene , C1 - C6 alkynyl, halogen , cyano , C1 - C6 alkyloxy, US 2020/0383331 A1 Dec. 10 , 2020 43
-continued ( R4 ) . X12 - X !! ( R4) HN— (CH2 ) ? HN -( CH2 ) , to o x ":/ = X ? X8 — X9 X3 NH (R ),
[ 0483 ] wherein a dashed line represents the presence or [ 0479 ] In one embodiments , R4 is OH . absence of a bond ; [ 0480 ] In some embodiments , compounds have a structure X8 - X12 is each independently selected from C , N , O , S ; selected from each R4 is independently H , OH , C1 - C6 alkyl, C1 - C6 alkylene, C1 - C6 alkynyl, halogen , cyano , C1 - C6 alkyloxy , aryl or heteroaryl each optionally substituted with a C1 - C6 OH OH , HN HN alkyl, SH , NHR3 , CO2R3 or halogen ; R3 is H , OH , C1 - C6 alkyl , C1 - C6 alkylene , C1 - C6 alkynyl, halogen , carboxylic group , ester group , aldehyde or cyano ; go p is 0 to 5 ; and NH2 q is 0 to 6 . [ 0484 ] In some embodiments, the compounds have a structure OH HN ( R4) p HN - (CH2 ) ?
NH
or X H X3 *( R2 ) OH . HN [ 0485 ] In still some embodiments, the compounds have a structure
NH (R4 ) , HN - ( CH2) ? 3 [ 0481 ] In some embodiments, the compounds have a structure , ( R2 ) , or (R4 ) p. XHN :/= x3 — R - Y HN— (CH2 ) ? 7 N X3 (R ). [ 0486 ] In some embodiments , the compound is meta wherein a dashed line represents the presence or absence of topolin , also known as 6- ( 3 -hydroxybenzylamino ) -purine , a bond . and by the abbreviation mT, having a empirical formula of [ 0482 ] In another embodiment, the compounds have a C12H10N5OH , a molecular weight of 241.25 , and the structure following structural formula : US 2020/0383331 A1 Dec. 10. 2020 44
[ 0490 ] In other embodiments, compounds include OH HN (R ) ( R) n
[ 0491 ] In one embodiment, the compound is thidiazuron , wherein said meta - topolin is a derivative of a willow tree or also known as 1 -phenyl -3- ( 1,2,3 - thiadiazol - 5 - yl )urea and a poplar tree. 5 -phenylcarbamoylamino - 1,2,3 - thiadiazole , has the empiri [ 0487 ] Meta - topolin analogues particularly include , with cal formula of C9H8N4OS , a molecular weight of 220.25 out limitation , meta - topolin riboside, meta -topolin -9 -gluco and the following structural formula side , ortho -topolin , ortho -topolin riboside, ortho -topolin - 9 glucoside , para - topolin , para - topolin riboside, para - topolin 9 - glucoside , ortho -methoxytopolin , ortho -methoxytopolin riboside , meta -methoxytopolin , meta -methoxytopolin ribo side and meta -methoxytopolin - 9 - glucoside. In particular embodiments, referred to herein as “ mT limited embodi ments ” , 6- ( 3 - fluorobenzylamino )purine ( FmT ) , 6-( 3 flurobenzylamino ) purine- 9 - riboside ( FmTR ) and / or 6- ( 3 [ 0492 ] Compounds useful according to the present disclo methoxybenzylamino )purine -9 - riboside (memTR ) can be sure include B - naphthoxyacetic analogues having a general excluded from the class of meta - topolin analogs. formula : [ 0488 ] Compounds useful according to the present disclo sure include thidiazuron analogues having a general formula xa Ra ( R ) (RS ) 16 ( R ) 14 X13* * Z2 or a salt thereof; wherein Ra is COR3 , CO2R3 , CONR3R4 , or CN ; wherein V is an aryl or heteroaryl; each Rb is independently R3 ; OR3 ; F ; Cl ; Br ; I ; CN ; NO2 ; each R5 and R6 is each independently H , OH , C1 - C6 alkyl, OCF3 ; CF3 ; NR2R3 ; SR3 , SOR3 , SO2R3 , CO2R3 , COR3 , C1 - C6 alkylene , C1 - C6 alkynyl, halogen , cyano , C1 - C6 CONR3R4 , CSNR4R5 ; or optionally substituted aryl or alkyloxy, aryl or heteroaryl each optionally substituted with optionally substituted heteroaryl, wherein each substituent a C1 - C6 alkyl or halogen ; of aryl or heteroaryl is independently C1 - C6 alkyl, F , CI , Br, or I ; n is 0 to 4 ; a is 1 , 2 , 3 , 4 , 5 , 6 , or 7 ; o is 0 to 5 X13 - X16 is each independently selected from C , N , O , S ; Xa is NH , S or O ; Z1 and Z2 are each independently NH , 0 , SH or CH or Z1 [ 0493 ] each R3 is independently H , C1 - C6 alkyl, C2 - C6 and Z2 can be combined to form a substituted or unsubsti alkenyl, or C2 - C6 alkynyl; and tuted aryl or heteroaryl; and each R4 is independently R3 or optionally substituted phe nyl, wherein each substituent of phenyl is independently Y1 is O or S. C1 - C6 alkyl, F , C1 , Br, or I. [ 0494 ] In another embodiment, compounds have a struc [ 0489 ] In another embodiment, compounds have a struc ture : ture .COH ( R" ) . ( Rba (RS ) n -16 Xx= t18 X14 to 413 N +2, [ 0495 ] In one embodiment, the compound is B -naph thoxyacetic acid (NAA ), also known as acetic acid , ( 2 -naph thalenoxy ) -( 9CI) and has a CAS Number of 120-23-0 , has wherein X17 - X21 is each independently selected from C , N , the empirical formula of C12H1003 , a molecular weight of O , S. 202.21 and the following structural formula : US 2020/0383331 A1 Dec. 10 , 2020 45
each R4 is independently R3 or optionally substituted phe CO2H nyl, wherein each substituent of phenyl is independently C1 - C6 alkyl, F , C1 , Br, or I. [ 0499 ] In another embodiment, compounds have a struc ture : [ 0496 ] Other examples of NAA analogues may include , but are not limited to : (R2 ) , CO2H , OH
CO2H , [ 0500 ] In one embodiment, the compound is indole butyric acid ( IBA ) , also known as 1 - Indole - 3 -butanoic acid , and has a CAS Number of 133-32-4 , has the empirical CI formula of C12H13NO2 , a molecular weight of 203.24 , and CO2H , the following structural formula:
CO2H , OH
F [ 0501 ] Other examples of IBA analogues may include, but H3CO CO2H , and are not limited to :
CO2H . CH3 , H3C [ 0497 ] Compounds useful according to the present disclo sure include indole butyric acid ( IBA ) analogues having a general formula : CH3, H3C -R (R2 )n H3C CH3 , H3C or a salt thereof; wherein R1 is COR3 , CO2R3 , CONR3R4 , or CN ; each R2 is independently R3 ; OR3 ; F ; Cl ; Br ; I ; CN ; NO2 ; OCF3 ; CF3 ; NR2R3 ; SR3 , SOR3 , SO2R3 , CO2R3 , COR3 , CH3 , CONR3R4 , CSNR4R5 ; or optionally substituted aryl or H3C optionally substituted heteroaryl, wherein each substituent CH3 of aryl or heteroaryl is independently C1 - C6 alkyl, F , C1 , Br, H3C or I ; n is 1 , 2 , 3 , or 4 ; X is NH , S or O ; CH3 , [ 0498 ] each R3 is independently H , C1 - C6 alkyl , C2 - C6 H3C NH alkenyl, or C2 - C6 alkynyl; and fore US 2020/0383331 A1 Dec. 10 , 2020 46
-continued -continued CH3 CH3 H3C , H3C
H3C H3C CH3, ?? , H3C H3C CH3
CH3 , ?? , sore CH3 CH3 OH , H3C
CH3 , soy H3C H3C OH , and CH3 H3C CH3, H3C CH3 ?? . H3Cfor [ 0502 ] Compounds useful according to the present disclo IZ CH3 , sure include benzylaminopurine ( BAP ) analogues having a H3C general formula : ( R5) OH , H3C
H3C (RO ??, or a salt thereof; H3C wherein a dashed line represents the presence or absence of a bond ; each R5 and each R6 is independently R3 ; OR3 ; F ; Cl ; Br ; I ; CN ; NO2 ; OCF3 ; CF3 ; NR2R3 ; SR3 , SOR3 , SO2R3 , ??, CO2R3 , COR3 , CONR3R4 , CSNR4R5 ; or optionally sub stituted aryl or optionally substituted heteroaryl, wherein H3C each substituent of aryl or heteroaryl is independently CH3 H3C C1 - C6 alkyl, F , C1 , Br, or I ; o is 0 , 1 , 2 , 3 , 4 , or 5 ; O p is 0 , 1 , or 2 ;
OH , X1 is NH, S or O ; H3C X4 is N = and X5 is —NH— , -S— , or O ; or X5 is —N— and X4 is —NH- , S— , or US 2020/0383331 A1 Dec. 10 , 2020 47
[ 0503 ] X2 and X3 and are independently N or CR6 ; each R3 is independently H , C1 - C6 alkyl, C2 - C6 alkenyl, or C2 - C6 alkynyl; and NH each R4 is independently R3 or optionally substituted phe nyl, wherein each substituent of phenyl is independently C1 - C6 alkyl, F , C1 , Br, or I. [ 0504 ] In some embodiments , X4 is N— and X5 is -NH , S or 0 , and the dashed line represents the presence or absence of a bond . Thus, compounds of according to the formula below are contemplated . [ 0509 ) Other examples of BAP analogues may include , but are not limited to : (RS ) NH 75
(RO ) Do NH [ 0505 ] In other embodiments, X5 is —N = and X4 is NH , S— , or 0. Thus, compounds of the formula below are contemplated .
Du?? (RS ) HN
D (R ) 13 -X4 ( 0506 ] In another embodiment, the compounds have a structure :
CodeNH (R5 ) NH
Oui( RO ) NH ( 0507 ] In another embodiment, the compounds have a structure :
RS NH NH or
RO NH [ 0508 ] In one embodiment, the compound is benzylami nopurine ( BAP ) , also known as 9H - Purin -6 - amine , N-( phe nylmethyl ) -, which has a CAS Number of 1214-39-7 , an empirical formula of C12H11N5 , a molecular weight of ca 225.25 , and the following structural formula : US 2020/0383331 A1 Dec. 10 , 2020 48
[ 0510 ] Compounds useful according to the present disclo sure include 6 - y -y- ( dimethylallylamino )-purine ( 2ip ) ana logues having a general formula : R8 X10
R8 X9 X10 ( R) my [ 0514 ] In other embodiments, X10 is -N = and X9 is NH , S , or0— . Thus , compounds having the (R ) structure shown below are contemplated . or a salt thereof; wherein a dashed line represents the presence or absence of a bond ; wherein R7 , R8 , and each R9 are independently R3 ; OR3 ; F ; R7 Cl ; Br ; I ; CN ; NO2 ; OCF3 ; CF3 ; NR2R3 ; SR3 , SOR3 , SO2R3 , CO2R3 , COR3 , CONR3R4 , CSNR4R5 ; or option ally substituted aryl or optionally substituted heteroaryl, R8 76 wherein each substituent of aryl or heteroaryl is indepen dently C1 - C6 alkyl, F , C1 , Br, or l ; * 7 810 qis 0 , 1 , or 2 ; X6 is NH , S or O ; (Rº ) X9 is —N — and X10 is —NH— , -S— , or 0— ; or X10 [ 0515 ] In another embodiment, compounds have a struc is —N— and X9 is —NH— , S or ture : [ 0511 ] X7 and X8 and are independently N or CR9 ; and each R3 is independently H , C1 - C6 alkyl, C2 - C6 alkenyl, or C2 - C6 alkynyl; and each R4 is independently R3 or optionally substituted phe nyl, wherein each substituent of phenyl is independently R7 C1 - C6 alkyl, F , C1 , Br, or I. [ 0512 ] In some embodiments, the dashed line represents the presence or absence of a bond . Thus, compounds of R8 NH according the formulas below are contemplated. N
R7 (R ) R8 [ 0516 ] In one embodiment, the compound is 6 - y ,y ,- (dim ethylallylamino ) -purine ( 2ip ) or DAP , also known as 9H -pu rin - 6 - amine , N- ( 3 -methyl - 2 -butene - 1 - yl) -, having a CAS (R ). No. 2365-40-4 , an empirical formula of C10H13N5 , a molecular weight of 203.24 , and the following structural formula : R8 X10 NH ( R ) .
[ 0513 ] In some embodiments X9 is –N = and X10 is -NH- , -S— , or O. Thus , compounds according the formula below are contemplated . US 2020/0383331 A1 Dec. 10 , 2020 49
[ 0517 ] Other examples of Zip analogues may include, but -continued are not limited to :
CI NH
NH
??? NH NH N N
NH NH
N and
NH
NH N
N [ 0518 ] Compounds useful according to the present disclo HO . sure include N , N - diphenylurea ( DPU ) analogues having a general formula : NH X11 X12
(R ' ), (R ! ), HO . or a salt thereof; NH wherein each R10 and each R11 is independently R3 ; OR3 ; F ; Cl ; Br ; I ; CN ; NO2 ; OCF3 ; CF3 ; NR2R3; SR3 , SOR3 , SO2R3 , CO2R3 , COR3 , CONR3R4 , CSNR4R5 ; or option ally substituted aryl or optionally substituted heteroaryl, wherein each substituent of aryl or heteroaryl is indepen dently C1 - C6 alkyl, F , C1 , Br, or 1 ; r and s are independently 0 , 1 , 2 , 3 , 4 , or 5 ; NH X11 and X12 are independently NR10 , S , or O ; each R3 is independently H , C1 - C6 alkyl, C2 - C6 alkenyl, or N C2 - C6 alkynyl; and each R4 is independently R3 or optionally substituted phe nyl , wherein each substituent of phenyl is independently C1 - C6 alkyl, F , C1 , Br, or I. US 2020/0383331 A1 Dec. 10 , 2020 50
[ 0519 ] In another embodiment, compounds have a struc ture : 7:13 4:14 ==? ( R12 ) ( R13 )
or a salt thereof; QYO(R19 ) , ( R !! ) , wherein each R12 and each R13 is independently R3 ; OR3 ; F ; Cl ; Br ; I ; CN ; NO2 ; OCF3 ; CF3 ; NR2R3; SR3 , SOR3 , SO2R3 , CO2R3 , COR3 , CONR3R4 , CSNR4R5; or option [ 0520 ] In one embodiment, the compound is N , N -diphe ally substituted aryl or optionally substituted heteroaryl, nylurea ( DPU ) , which is represented by a formula : wherein each substituent of aryl or heteroaryl is indepen dently C1 - C6 alkyl, F , C1 , Br, or I ; t and u are independently 0 , 1 , 2 , 3 , 4 , or 5 ; X13 and X14 are independently NR12 , S , or O ; each R3 is independently H , C1 - C6 alkyl, C2 - C6 alkenyl, or OYU C2 - C6 alkynyl; and each R4 is independently R3 or optionally substituted phe nyl, wherein each substituent of phenyl is independently [ 0521 ] Other examples of DPU analogues may include, C1 - C6 alkyl, F , Cl , Br, or I. but are not limited to : [ 0523 ] In one embodiment, compounds have a structure :
CI, 413 X14 .Ci .
OYOTI (R12 ) ( R13) u- 1 ZI [ 0524 ] In another embodiment, compounds have a struc CI, ture :
Ci.
( R ! ), (R13 ) -1 [ 0525 ] In one embodiment, the compound is N-( 2 -chloro pyridin - 4 - yl) -N '- phenylurea , which is represented by a for OYO mula : F , or OYO Ci.
[ 0526 ] Other examples of PPU analogues may include , but oyn are not limited to : OCH3.
[ 0522 ] Compounds useful according to the present disclo Br, sure include N -pyridinyl - N - phenylurea ( PPU used inter changeably with CPPU herein ) analogues having a general formula : US 2020/0383331 A1 Dec. 10 , 2020 51
sample may be increased from one to hundreds or thousands -continued of plants. Depending on the type of tissue grown , multipli cation can involve different methods and media . If the plant material grown is callus tissue , it can be placed in a blender N , and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue . If the tissue is grown as small plants called plantlets , hormones are often added that cause the plantlets to produce many small off shoots that can be removed and recultured . N, [ 0531 ] The next stage ( pretransplant ” stage ) involves treating the plantlets / shoots produced to encourage root growth and “ hardening. ” It is performed in vitro , or in a sterile or substantially sterile environment. “ Hardening ” refers to the preparation of the plants for a natural growth environment. Until this stage , the plantlets have been grown in “ ideal” conditions, designed to encourage rapid growth . F , or Due to lack of necessity, the plants are likely to be highly susceptible to disease and often do not have fully functional dermal coverings and will be inefficient in their use of water and energy . In vitro conditions are high in humidity and plants grown under these conditions do not form a working cuticle and stomata that keep the plant from drying out, N. when taken out of culture the plantlets need time to adjust to more natural environmental conditions. Hardening typically involves slowly weaning the plantlets from a high -humidity , OCH3 low light, warm environment to what would be considered a normal growth environment for the species in question. [ 0532 ] In the final stage of plant micropropagation , the Propagation /Micropropagation plantlets are removed from the plant media and transferred ( 0527 ] Micropropagation is the practice of rapidly multi to soil or (more commonly ) potting compost for continued plying stock plant material to produce a large number of growth by conventional methods. This stage is often com progeny plants, using modern plant tissue culture methods. bined with the “ pretransplant” stage . Micropropagation is used to multiply novel plants , such as [ 0533 ] Modern plant tissue culture is performed under those that have been genetically modified or bred through aseptic conditions under filtered air . Living plant materials conventional plant breeding methods . It is also used to from the environment are naturally contaminated on their provide a sufficient number of plantlets for planting from a surfaces ( and sometimes interiors ) with microorganisms, so stock plant which does not produce seeds , or does not surface sterilization of starting materials ( explants ) in respond well to vegetative reproduction . chemical solutions (usually alcohol or bleach ) is required. [ 0528 ] Micropropagation can first begin with the selection Explants are then usually placed on the surface of a solid of plant material to be propagated. Clean stock materials that culture medium , but are sometimes placed directly into a are free of viruses and fungi are important in the production liquid medium , particularly when cell suspension cultures of the healthiest plants . are desired . Solid and liquid media are generally composed [ 0529 ] Once the plant material is chosen for culture , the of inorganic salts plus a few organic nutrients, vitamins and collection of explant ( s) begins and is dependent on the type plant hormones . Solid media are prepared from liquid media of tissue to be used , including stem tips , anthers , petals , with the addition of a gelling agent, usually purified agar . pollen and others plant tissues . The explant material is then [ 0534 ] The composition of the medium , particularly the surface sterilized , usually in multiple courses of bleach and plant hormones and the nitrogen source (nitrate versus alcohol washes and finally rinsed in sterilized water . This ammonium salts or amino acids ) have profound effects on small portion of plant tissue , sometimes only a single cell , is the morphology of the tissues that grow from the initial placed on a growth medium , typically containing sucrose as explant. For example , an excess of auxin will often result in an energy source and one or more plant growth regulators a proliferation of roots, while an excess of cytokinin may ( plant hormones ). Usually the medium is thickened with yield shoots . A balance of both auxin and cytokinin will agar to create a gel which supports the explant during often produce an unorganized growth of cells , or callus , but growth . Some plants are easily grown on simple media but the morphology of the outgrowth will depend on the plant others require more complicated media for successful species as well as the medium composition . As cultures growth ; the plant tissue grows and differentiates into new grow , pieces are typically sliced off and transferred to new tissues depending on the medium . For example, media media ( subcultured ) to allow for growth or to alter the containing cytokinins are used to create branched shoots morphology of the culture . The skill and experience of the from plant buds . tissue culturist are important in judging which pieces to [ 0530 ] Multiplication is the taking of tissue samples pro culture and which to discard . As shoots emerge from a duced during the first stage and increasing their number. culture , they may be sliced off and rooted with auxin to Following the successful introduction and growth of plant produce plantlets which , when mature , can be transferred to tissue , the establishment stage is followed by multiplication . potting soil for further growth in the greenhouse as normal Through repeated cycles of this process , a single explant plants. US 2020/0383331 A1 Dec. 10 , 2020 52
[ 0535 ] The tissue obtained from the plant to culture is [ 0543 ] 5. To cross -pollinate distantly related species and called an explant. Based on work with certain model sys then tissue culture the resulting embryo, which would oth tems , particularly tobacco , it has often been claimed that a erwise normally die (Embryo Rescue ). totipotent explant can be grown from any part of the plant. [ 0544 ] 6. For production of doubled monoploid ( dihap However, this concept has been vitiated in practice . In many loid ) plants from haploid cultures to achieve homozygous species explants of various organs vary in their rates of lines more rapidly in breeding programs, usually by treat growth and regeneration, while some do not grow at all . The ment with colchicine which causes doubling of the chromo choice of explant material also determines if the plantlets some number. developed via tissue culture are haploid or diploid . Also the [ 0545 ] 7. As a tissue for transformation , followed by either risk of microbial contamination is increased with inappro short - term testing of genetic constructs or regeneration of priate explants. Thus it is very important that an appropriate transgenic plants . choice of explant be made prior to tissue culture . [ 0546 ] 8. Certain techniques such as meristem tip culture [ 0536 ] The specific differences in the regeneration poten can be used to produce clean plant material from infected tial of different organs and explants have various explana stock , such as potatoes and many species of soft fruit. tions . The significant factors include differences in the stage [ 0547 ] 9. Micropropagation using meristem and shoot of the cells in the cell cycle , the availability of or ability to culture to produce large numbers of identical individuals. transport endogenous growth regulators, and the metabolic [ 0548 ] Micropropagated plants can begin from a selected capabilities of the cells . The most commonly used tissue piece of plant tissue , called an “ explant ” or “ mother plant. ” explants are the meristematic ends of the plants like the stem This explant is the source of cells to be developed during the tip , auxiliary bud tip and root tip . These tissues have high tissue culturing process . For example, the explant can be any rates of cell division and either concentrate or produce segment or collection of cells from apical meristems, ter required growth regulating substances including auxins and minal buds , axillary buds , adventitious buds , accessory cytokinins. Some explants , like the root tip , are hard to buds , pseudo - terminal buds , cambium , lateral meristem , isolate and are contaminated with soil microflora that lateral bud , vegetative buds , reproductive buds , mixed buds , become problematic during the tissue culture process. Cer shoot segments , shoot apices , stem segments , immature tain soil microflora can form tight associations with the root nodal sections from stems, lateral shoots , seedlings, seeds , systems , or even grow within the root. Soil particles bound shoots starting to rise from the ground, immature flower to roots are difficult to remove without injury to the roots buds , inflorescences, crown segments, leaf segments, or any that then allows microbial attack . These associated micro part thereof. In one embodiment, the explant is taken from flora will generally overgrow the tissue culture medium a plant of about 1 week old , about 2 weeks old , about 3 before there is significant growth of plant tissue . Aerial weeks old , about 1 month old , about 2 month old , about 3 ( above soil ) explants are also rich in undesirable microflora . months old , about half year old , about 1 year old , about 2 However , they are more easily removed from the explant by years old plant, about 3 years old , about 5 years old , or more . gentle rinsing , and the remainder usually can be killed by The plant from which the explant is obtained can be grown surface sterilization . Most of the surface microflora do not in any suitable conditions , including but not limited to form tight associations with the plant tissue . Such associa growing in a growth chamber, growing in a greenhouse , tions can usually be found by visual inspection as a mosaic , growing in a field, or growing in a tissue culture container de - colorization or localized necrosis on the surface of the ( petri dish , margenta box , etc. ). In some embodiments, the explant. explant is tissue culture obtained from shoot clumps main [ 0537 ] An alternative for obtaining uncontaminated tained as stock on growth media . In some embodiments , the explants is to take explants from seedlings which are asep explant is a nodal section having one or more axillary bud , tically grown from surface - sterilized seeds . The hard surface which can be dormant or active . In some other embodi of the seed is less permeable to penetration of harsh surface ments, the explant is a seed or a part thereof. sterilizing agents , such as hypochlorite, so the acceptable [ 0549 ] In some embodiments, the tissue culture is conditions of sterilization used for seeds can be much more obtained from shoot clumps maintained on growth media as stringent than for vegetative tissues . stock . In some embodiments, the explant is a segment of [ 0538 ] Tissue cultured plants are clones , if the original bamboo cane . In some embodiments, the segment of bam mother plant used to produce the first explants is susceptible boo cane comprises an internode. In some embodiments, the to a pathogen or environmental condition , the entire crop segment of bamboo cane comprises a nodal section . In some would be susceptible to the same problem , and conversely embodiments, the nodal section comprises a single bud . In any positive traits would remain within the line also . Plant some embodiments, the bud is dormant or active . In some tissue culture is used widely in plant science ; it also has a embodiments, the explant is taken from a plant of about 1 number of commercial applications. Applications include : week old , about 2 weeks old , about 3 weeks old , about 1 [ 0539 ] 1. Micropropagation is widely used in forestry and month old , about 2 month old , about 3 months old , about in floriculture . Micropropagation can also be used to con half year old , about 1 year old , about 2 years old plant, about serve rare or endangered plant species . 3 years old , about 5 years old , or more . In some embodi [ 0540 ] 2. A plant breeder may use tissue culture to screen ments, a bamboo seed or a part thereof is used . cells rather than plants for advantageous characters, e.g. [ 0550 ] Availability of virus free starting material is desir pathogen resistance / tolerance . able for an agricultural seed production program . Thus, in [ 0541 ] 3. Large - scale growth of plant cells in liquid cul some embodiments, the virus - free micropropagated plants ture inside bioreactors as a source of secondary products , begin from an explant that is subjected to one or more like recombinant proteins used as biopharmaceuticals. antiviral treatments , such as a chemical antiviral, thermo [ 0542 ] 4. To cross distantly related species by protoplast therapy, and / or meristem - tip culture . Meristem culture is one fusion and regeneration of the novel hybrid . procedure used to produce a virus - free plant. In this method , US 2020/0383331 A1 Dec. 10 , 2020 53 apical or axillary growing tips ( 0.1-0.3 mm ) are dissected induced and regenerated from the above cultures using and allowed to grow into plantlets on special culture either organogenesis or somatic embryogenesis. medium under controlled conditions . The meristem culture [ 0555 ] In some embodiments , the tissue culture is for virus elimination is based on the principle that many obtained from shoot clumps maintained on growth media as viruses are unable to infect the apical/ axillary meristem of a stock . In some embodiments, the explant is a segment of growing plant and that a virus - free plant can be produced if bamboo cane . In some embodiments, the segment of bam a small ( e.g. 0.1-0.3 mm ) piece of meristemic tissue is boo cane comprises an internode . In some embodiments , the propagated. Excision of very small meristems typically segment of bamboo cane comprises a nodal section . In some requires a high degree of expertise and the development of embodiments, the nodal section comprises a single bud . In plants from these small meristems (mericlones ) can be some embodiments, the bud is dormant or active . In some lengthy ( i.e. 4 to 8 months ). To increase the percentage of embodiments, the explant is taken from a plant of about 1 virus freedom in regenerated mericlones , meristem culture week old , about 2 weeks old , about 3 weeks old , about 1 can be combined with other antiviral treatments , such as month old , about 2 month old , about 3 months old , about thermotherapy ( high temperature treatment) or chemo half year old , about 1 year old , about 2 years old plant, about therapy ( treatment with antiviral compounds ) to increase the 3 years old , about 5 years old , or more . In some embodi production of virus - free plants . ments, a bamboo seed or a part thereof is used . [ 0551 ] Thus, in some embodiments, the method comprises [ 0556 ] The present invention provides methods for in vitro using meristem culture, thermotherapy, chemotherapy , or micropropagation of plant, for example, gymnosperm any combination thereof to produce a virus - free plant. In one plants, angiosperm plants, monocot plants, dicot plants, embodiment, meristem culture , thermotherapy, and chemo crops, agriculturally / economically / environmentally impor therapy, are used to produce a virus - free plant. In some tant plants, etc. embodiments , the use of an antiviral can increase the success [ 0557 ] The present invention provides methods for in vitro in producing a virus - free plant by at least two or three times . micropropagation of plant, for example , gymnosperm In one embodiment, chemotherapy comprises using an anti plants, angiosperm plants, monocot plants, dicot plants, viral in a medium to culture the explant. In another embodi crops , agriculturally / economically / environmentally impor ment, thermotherapy comprises incubating an explant under tant plants, etc. a 16 h light photoperiod at 30-40 umol / m2 / s light intensity at [ 0558 ] In some embodiments, the methods disclosed 37 ° C. In some embodiments, the thermotherapy is for one herein can be used for in vitro micropropagation of a week . gymnosperm plant . For example, the methods can be used [ 0552 ] Accordingly, in one aspect of the present invention , for in vitro micropropagation of the plants in the family ! a method for producing a virus - free plant comprises incu order of Cycadaceae , Zamiaceae , Ginkgoaceae , Welwitschi bating an explant with medium , optionally comprising an aceae , Gnetaceae , Ephedraceae , Pinaceae, Araucariaceae, antiviral; optionally, subjecting an explant of the plant Podocarpaceae, Sciadopityaceae, Cupressaceae , or Tax culture to thermotherapy, wherein the explant grows into a aceae .. plantlet; excising an apical meristem from the plantlet; and [ 0559 ] In some embodiments, the methods disclosed placing the apical meristem into a regeneration media ; herein can be used for in vitro micropropagation of an wherein a virus - free plantlet is produced . The excision is of angiosperm plant. For example , the methods can be used for a very small piece of meristem , and can be performed as in vitro micropropagation of the plants in the family order of depicted in FIG . 33 . Ceratophyllum , Chloranthaceae , eudicots, magnoliids, or [ 0553 ] In some embodiments, the regeneration media is monocots . selected from FIG . 32. In other embodiments, the regenera [ 0560 ] In some embodiments , the methods disclosed tion media comprises an antiviral, such as Ribavirain ( also herein can be used for in vitro micropropagation of a dicot known as Virazole ). The method for producing a virus - free plant. For example , the methods can be used for in vitro plant can also comprise culturing or subculturing, using micropropagation of the plants in the family / order of Bux conditions such as disclosed in PCT Publication No. aceae , Didymelaceae , Sabiaceae , Trochodendraceae , Tetra WO2013016198 , which is incorporated by reference in its centraceae , Ranunculales , Proteales , Aextoxicaceae , Ber entirety. For example, culturing or subculturing can be of the beridopsidaceae , Dilleniaceae , Gunnerales, Caryophyllales, explant, apical meristem , the plantlet, or any combination Saxifragales, Santalales , rosids , Aphloiaceae, Geissolomata thereof. The culturing or subculturing can be performed ceae , Ixerbaceae, Picramniaceae , Strassburgeriaceae , Vita every one , two to three weeks . In one embodiment, culturing ceae , Crossosomatales, Geraniales, Myrtales, Zygophyl comprises incubating the explant under a 16 h light photo laceae , Krameriaceae, Huaceae , Celastrales, Malpighiales, period at 80-100 umol/ m² / x light intensity at 24 ° C. In some Oxalidales , Fabales , Rosales , Cucurbitales, Fagales , Tapi embodiments, the method for producing a virus - free plant sciaceae , Brassicales , Malvales , Sapindales, asterids, Cor uses one or more different regeneration media , such as nales , Ericales , Boraginaceae, Icacinaceae , Oncothecaceae , depicted in FIG . 32 . Vahliaceae, Garryales, Solanales , Gentianales, Lamiales, ( 0554 ] The plantlet produced by a method disclosed herein Bruniaceae , Columelliaceae , Desfontainiaceae, Eremosyn can be subcultured or tested for viruses . Any method known aceae , Escalloniaceae , Paracryphiaceae , Polyosmaceae , for testing for the presence of a virus can be used , such as Sphenostemonacae , Tribelaceae , Aquifoliales, Apiales, Dip by enzyme- linked immunosorbent assay ( ELISA ) . The sacales , or Asterales . plantlet can be multiplied and subcultured , and used for [ 0561 ] In some embodiments, the methods disclosed further propagation . The pssent invention is applicable to a herein can be used for in vitro micropropagation of a whole range of agricultural crops where a protocol for monocot plant. For example , the methods can be used for in isolation and culture of meristematic cells or meristematic vitro micropropagation of the plants in the family order of zones in vitro are available . Plantlets could be further Acorales , Alismatales , Asparagales , Dioscoreales , Liliales , US 2020/0383331 A1 Dec. 10 , 2020 54
Pandanales, Petrosaviales, Dasypogonaceae , Arecales , analog thereof. In some embodiments , the bud induction Commelinales, Poales , or Zingiberales. medium ( B001 ) comprises only one strong cytokinin , [ 0562 ] In some embodiments, the methods disclosed wherein the cytokinin is TDZ or analog thereof. In some herein can be used for in vitro micropropagation of a embodiments, the concentration of the strong cytokinin bamboo species , such as Phyllostachys edulis ( e.g. , Phyl ( e.g. , TDZ ) in the bud induction media ( B001 ) is about 0.25 lostachys edulisi Moso ') , Phyllostachys bissetti, Fargesia mg / L to about 100 mg / L , for example , about 0.5 mg / L to denudata , Pleioblastus fortunei, Sasa Veitchii, Pleioblastus about 2 mg / L . viridistriatus, Thamnocalamus crassinodus, Chusquea [ 0568 ] Examples of a shoot elongation /maintenance Culeo “ Cana Prieta " , Bambusa Old Hamii , Phyllostachys media ( BOO2 ) are described herein . In some embodiments , Moso, Phyllostachys Atrovaginata , Dendrocalamus Asper , a shoot elongation /maintenance medium ( B002 ) comprises or Guadua Angustifolia . In some embodiments, the bamboo one or more cytokinin that is relatively weaker cytokinin , species is Phyllostachys edulis, Moso . such as a cytokinin other than TDZ . In some embodiments , [ 0563 ] In some embodiments , the plant is a non - bamboo the shoot elongation /maintenance medium ( B002 ) com species . In some embodiments , the non -bamboo plant spe prises only one relatively weaker cytokinin , such as BAP, cies is Geranium spp . ( e.g. , Geranium rozanne ), meta - topolin , ip ( e.g. , 2ip ) , zeatin , zeatin riboside , or com Hakonechloa macra ( e.g. , Hakonechloa macra ' Aureola ’, bination thereof. In some embodiments , the shoot elonga Hakonechloa macra ‘ All gold ' ) , Helleborus ( e , g . , Hellebo tion /maintenance medium ( BOO2 ) comprises more than one rus ‘ Ivory Prince ” ) , Phormium , Wasabi ( e.g. , Wasabi C2 ) , cytokinins. In some embodiments, the concentration of a Arundinaria ( e.g. , Arundinaria gigantean ) , or Solanum ( e.g. , cytokinin in a shoot elongation /maintenance medium Solanum tuberosum and Solanum tuberosum ). ( BOO2 ) is about 0.01 mg / L to about 100 mg/ L , for example , [ 0564 ] In some embodiments , the methods are used for 0.25 mg / L to about 5 mg / L . rapid bamboo in vitro micropropagation . High shoot multi [ 0569 ] In some embodiments , the bud induction medium plication rate can be achieved in the methods disclosed ( B001 ) and / or the shoot elongation /maintenance medium herein . As used herein , the phrase “ multiplication rate ” ( B002 ) comprises one or more auxin , such as ß -naphthoxy refers to the multiplication fold of plant shoots obtained in acetic acid (NAA ), 2,4 - Dichlorophenoxy acetic acid (2,4 a micropropagation process by starting from a single D ) , indole - 3 - butyric acid ( IBA ) , indole - 3 - acetic acid ( IAA ), explant. For example , in the situation where the explant is a picloram , or analogs thereof . In some embodiments , the bud nodal section comprising a single bud , and 3 shoots are induction medium ( B001 ) and / or the shoot elongation / obtained after a micropropagation cycle, the multiplication maintenance medium (B002 ) comprises NAA . In some rate is 3x . In some embodiments, by using the bud induction embodiments, the concentration of an auxin in the media is media ( B001 ) and the shoot elongation /maintenance media 0.01 mg / L to about 50 mg/ L , for example, about 0.25 mg / L ( BOO2 ) , a multiplication rate of at least about 2x to about to about 0.5 mg / L . 30x can be achieved after micropropagation . For example, [ 0570 ] In some embodiments, the methods are used for about 2x , 3x , 4x , 5x , 6x , 7x , 8X , 9X , 10x , 11x , 12x , 13x , micropropagating plants in vitro . In some embodiments , the 14x , 15x , 16x , 17 , 18 , 19 , 20 , 21x , 22x , 23x , 24x , 25x , methods comprise ( a ) incubating a plant tissue culture , 26x , 27x , 28x , 29x , about 30x , or more can be achieved explant or seed in a first medium , and ( b ) then incubating the within about 5 days , 6 days, 7 days, 8 days, 9 days , 10 days , plant tissue obtained from step ( a ) in a second medium . In 11 days , 12 days , 13 days , 14 days , 15 days, 16 days, 17 some embodiments, the first medium is a bud induction days, 18 days, 19 days , 20 days, 21 days , 22 days, 23 days , medium ( B001 ) , and the second medium is shoot elon 24 days, 25 days, 26 days , 27 days , or about 28 days , or gation and maintenance medium ( BOO2 ) . more . [ 0571 ] In some embodiments , the methods comprise ( a ) [ 0565 ] In some embodiments , the present invention is incubating a tissue culture, explant or seed / seed part in a bud based on the unexpected discovery that a pulsed treatment of induction medium ( B001 ) to induce shoot bud formation ; an explant on a first medium comprising a strong cytokinin , ( b ) incubating the shoot buds obtained in step ( a ) in a shoot such as TDZ , followed by a treatment of the explant on a elongation /maintenance medium ( B002 ) . second medium comprising one or more cytokinins other [ 0572 ] The methods can further comprise ( c ) incubating than TDZ , e.g. , cytokinins that are relatively weaker than the shoots from step ( b ) in a bud induction medium ( B001 ) TDZ , such as meta - topolin , kinetin , isopentenyl adenine ( iP , to induce shoot bud formation ; and ( d ) incubating the shoot e.g. , 2ip ) , zeatin , trans - zeatin , zeatin riboside, dihydrozeatin , buds obtained in step ( c ) in a shoot elongation /maintenance benzyleadenin ( BAP ) , or benzyladenosine ( [ 9R ] BAP ) , can medium ( B002 ) . provide rapid in vitro micropropagation with unexpected [ 0573 ] In some embodiments, the shoot buds obtained in high multiplication rate . step ( a ) and / or step ( c ) are separated prior to incubating the [ 0566 ] In some embodiments , the methods comprise using shoot buds in step ( b ) and / or step ( d ) . In some embodiments , a bud induction medium ( B001 ) and a shoot elongation / the separation produces groups of 1 to 3 shoot buds per maintenance media ( BOO2 ) , wherein the bud induction separation prior to incubating the shoot buds in step ( b ) medium ( B001 ) comprises a strong cytokinin , such as and / or step ( d ). TDZ , and the shoot elongation /maintenance medium [ 0574 ] In some embodiments , the methods further com ( B002 ) comprises a relatively weaker cytokinin , such as prises ( e ) repeating the incubating steps ( c ) and step ( d ) for meta - topolin , kinetin , isopentenyl adenine ( iP , e.g. , 2ip ) , at least once . zeatin , trans - zeatin , zeatin riboside, dihydrozeatin , benzy [ 0575 ] In some embodiments, when a bud induction leadenin ( BAP ) , or benzyladenosine ( [ 9R ] BAP ) . medium ( B001 ) and a shoot elongation and maintenance [ 0567 ] Examples of a bud induction medium ( B001 ) are medium ( B002 ) are used , the methods further comprise: ( e ) described herein . In some embodiments , a bud induction repeating the incubating step ( c ) and step ( d ) for at least one , medium ( B001 ) comprises one or more strong cytokinin or at least two , at least three, at least four, at least five , at least US 2020/0383331 A1 Dec. 10 , 2020 55 six , at least seven , at least eight, or more additional cycles . 15 , at least 20 , at least 25 , at least 26 , at least 27 , at least 28 , There is no limit to how many times the cycling of step ( c ) at least 30 , or more shoot buds for each bamboo tissue and step ( d ) can be repeated . Buds and / or shoots obtained in culture, explant or seed placed in the bud induction medium step ( a ) or step ( c ) can be separated prior to the buds and / or ( B001 ) of step ( a ) or ( c ) . shoots entering step ( b ) or step ( d ), respectively, wherein [ 0580 ] In some embodiments, the incubation period of such separation can result in a single bud or shoot , 2 buds step ( a ) or step ( c ) lasts for about one hour to about three and / or shoots , 3 buds and / or shoots , or 4 or more buds and / or weeks, or more . For example, the incubation period of step shoots per separation . Optimum separation for maximum , ( a ) or step ( c ) lasts for about 1 hour, about 2 hours, about 3 rapid production of bamboo copies of a single species , hours , about 4 hours, about 6 hours , about 7 hours , about 8 genotype or clone usually involves separating the buds hours , about 9 hours, about 10 hours, about 11 hours , about and / or shoots obtained in step ( a ) or step ( c ) into 1-3 buds 12 hours, about 14 hours , about 16 hours , about 18 hours, and / or shoots prior to entering into step ( b ) or step ( d ) , about 20 hours, about 22 hours , about 24 hours, about 30 respectively . Where there are 2 or more buds and / or shoots hours , about 36 hours , about 42 hours , about 48 hours , about per separation this is known in the art as a clumping or 54 hours, about 60 hours, about 1 week , about 1.5 weeks, “ clump” of buds and / or shoots . Some variation in the about 2 weeks, about 2.5 weeks , about 3 weeks, about 3.5 methodologies of the present invention may be necessary so weeks, about 4 weeks, or more . In some embodiments, the as to fine - tune the process for specific species , genotypes or incubation of step ( a ) and / or step ( c ) lasts from about 24 clones of bamboo and such process variations are within the hours to about 60 hours . Thus, the incubation of step ( a ) disclosure of this invention . and / or step ( c ) can last for about 24 hours , about 25 hours , [ 0576 ] In some embodiments , both the bud induction about 26 hours, about 27 hours , about 28 hours, about 29 medium ( B001 ) and the shoot elongation /maintenance hours , about 30 hours , about 35 hours , about 40 hours , about medium ( BOO2 ) are liquid media . The advantage of liquid 45 hours, about 50 hours , about 55 hours , about 56 hours, media is that the one can replace old media with fresh media , about 57 hours, about 58 hours , about 59 , hours, or about 60 or replace one type of media with another type of media hours . In some embodiments the incubation stage of step ( a ) quickly and easily, without transferring the seedlings of or step ( c ) can last longer than 60 hours , e.g. , about 1 week , plant from one container to another. Therefore , in some about 2 weeks , about 3 weeks , about 4 weeks , or longer. embodiments, the whole micropropagation process is [ 0581 ] In some embodiments , the incubation of step ( b ) achieved in a single container, for example, in a bioreactor. and / or step ( d ) lasts for any desired period . In some embodi [ 0577 ] In some embodiments, the bud induction media ments, the incubation of step ( b ) and / or step ( d ) lasts from ( B001 ) and / or the shoot elongation /maintenance media about 24 hours to about four weeks , or more . For example, ( BOO2 ) are semi- solid or solid media . In some embodi the incubation of step ( b ) and / or step ( d ) lasts from about ments , liquid media and semi - solid or solid media can be three days to about five days, or more . Thus, the incubation used subsequently with any desired order . For example , the of step ( b ) and / or step ( d ) can last for about 24 hours , about bud induction medium (B001 ) in step ( a ) and / or step ( c ) is 25 hours, about 26 hours , about 27 hours, about 28 hours, liquid, semi - solid , or solid ; the shoot elongation /mainte about 29 hours, about 30 hours , about 35 hours , about 40 nance medium ( BOO2 ) in step ( b ) and / or step ( d ) is liquid , hours , about 45 hours , about 48 hours , about 50 hours , about semi- solid , or solid . Thus , in some embodiments, the bud 55 hours, about 56 hours , about 57 hours , about 58 hours, induction medium ( B001 ) of step ( a ) and / or step ( c ) is a about 59 , hours, about 60 hours , about 72 hours, about 96 liquid medium . In some embodiments, the bud induction hours or about 120 hours . In some embodiments the incu medium ( B001 ) of step ( a ) and / or step ( c ) is a solid bation of step ( b ) and / or ( d ) can last longer than 120 hours , medium . In some embodiments, the shoot elongation /main e.g. , about 1 week , about 2 weeks, about 3 weeks , about 4 tenance medium ( B002 ) of step ( b ) and / or step ( d ) is a weeks, or longer. In some embodiments, the incubation liquid medium . In some embodiments , the shoot elongation / period of step ( b ) or step ( d ) lasts for about 0.5 week , about maintenance media ( BOO2 ) of step ( b ) and / or step ( d ) is a 1 week , about 1.5 weeks, about 2 weeks, about 2.5 weeks, solid media . about 3 weeks, about 3.5 weeks , about 4 weeks, about 4.5 [ 0578 ] In some embodiments the methods of the present weeks, about 5 weeks , about 5.5 weeks , about 6 weeks, inventionmay involve using a liquid media for one step and about 7 weeks , about 8 weeks , or more . a solid media for the next step of a particular cycle . For [ 0582 ] The incubation periods in the steps can be adjusted example , the present invention encompasses methods depending on the species of the plant, type of the explant, a whereby step ( a ) is accomplished using liquid media and desired multiplication rate . Without wishing to be bound by step ( b ) is accomplished using solid media . Alternatively, if any theory, in some embodiments , for a bamboo species , the both steps ( a ) and ( b ) and / or steps ( c ) and ( d ) are both done incubation period in step ( a ) or step ( c ) can be about 1 hour using liquid media , then the present invention contemplates to about 3 weeks , for example , about 24 hours to about 60 that the liquid media may be changed without moving the hours ; and the incubation period in step ( b ) or step ( d ) can buds and / or shoots to another container ( e.g. , test tube , be about 24 hours to about 4 weeks , for example , about 3 bioreactor, jar, etc. ) . For example, if both steps ( a ) and ( b ) days to about 5 days . are accomplished using liquid media in a hydroponic setup , [ 0583 ] The shoot multiplication rate can be further then the buds and / or shoots may remain in their fixed or improved by repeating step ( c ) and step ( d ) . For example , the unfixed position while the liquid media is replaced. multiple shoots developed after treatment of step ( a ) and [ 0579 ] In some embodiments, the incubation of step ( a ) treatment of step ( b ) can be subjected to one or more round and / or step ( c ) lasts for a period that is sufficient to produce of treatment of step ( c ) and treatment of step ( d ). In some more than one shoot bud . For example, the period is set so embodiments , treatment in step ( c ) and treatment of step ( d ) as to produce at least 1 , at least 2 , at least 3 , at least 4 , at least are conducted at least once , at least twice , at least three 5 , at least 6 , at least 7 , at least 8 , at least 9 , at least 10 , at least times , at least four times , at least five times , at least six US 2020/0383331 A1 Dec. 10 , 2020 56 times , at least seven times , at least eight time , or more . Since or red clover. In some embodiments, it is used for micro the treatments in all steps are in short periods, a very short propagation of a grass . The grass can be of the Poaceae ( or total time is needed to reach a very high shoot multiplication Gramineae ), Cyperaceae or Juncaceae family. The grass can rate . be a perennial grass or a cereal grass. The grass can be [ 0584 ] In some embodiments , each step of ( a ) to ( e ) can switchgrass , big bluestem , miscanthus, alfalfa, orchard also be repeated before conducting the next step , by replac grass, or reed canarygrass . The grass can be bamboo , tall ing old media with fresh media , for once , twice, three times , fescue , goat grass , or Kentucky bluegrass . or more . In some embodiments, step ( e ) is repeated at least [ 0590 ] Other types of grasses include wheat, rye , oat, once , twice , three times , or more . In some embodiments, barley, soy, and hemp, as well as straws derived therefrom . steps ( a ) to ( e ) take approximately one week , two weeks , In some embodiments, it is used for micropropagation of a three weeks , four weeks , five weeks , six weeks , or more . phyto - pharmaceutical plant. In some embodiments, it is used [ 0585 ] In some embodiments , starting from a single for micropropagation of Aloe vera , Ginger, Grape , Canna explant, the present methods can provide about 10x to about bis, Garlic, Onion , Echinacea , Geranium , Hakonechloa , 30x shoot multiplication rate in approximately three weeks . Miscanthus, Arundo donax , Switch grass , Rice , or Sugar In addition , at least about 500 , at least about 1,000 , at least cane . about 2,000 , at least about 3,000 , at least about 4,000 , at [ 0591 ] Referring again to the system 100 in FIG . 5 , in use least about 5,000 , at least about 6,000 , at least about 7,000 , for plant micropropagation , the plant propagation sequence at least about 8,000 , at least about 9,000 , at least about starts with placing an explant into the growth vessel 110. In 10,000 , at least about 20,000 , at least about 30,000 , at least some embodiments, the first media container 130 comprises about 40,000 , at least about 50,000 , at least about 60,000 , at a bud induction medium ( B001 ) as described herein , and least about 70,000 , at least about 80,000 , at least about the second media container 150 comprises a shoot elonga 90,000 , at least about 100,000 , or more plant shoots can be tion /maintenance medium ( BOO2 ) . obtained within about 6 weeks , about 10 weeks , about 2 [ 0592 ] In some embodiments, the bud induction medium months, about 2.5 months, about 3 months, about 4 months, ( BO01 ) comprises an effective amount of thidiazuron about 5 months, about 6 months . ( TDZ ) or analog thereof, and wherein the shoot elongation / [ 0586 ] To further improve the shoot multiplication rate , a maintenance medium ( BOO2 ) comprises an effective separation step can be added during or immediately after one amount of one or more cytokinins other than TDZ or an or more steps selected from steps ( a ) , ( b ), ( c ) , and ( d ). For analog thereof. In some embodiments, the concentration of example, multiple shoot buds produced in step ( a ) and / or TDZ or analog thereof in the bud induction medium ( B001 ) step ( c ) , or multiple shoots produced in step ( c ) and / or step is about 0.25 mg / L to about 100 mg / L , e.g. , from 0.5 mg/ L ( c ) can be separated into individual pieces , and each of the to about 2 mg / L . In some embodiments, one or more separated pieces can be placed in an individual container cytokinins other than TDZ or an analog thereof in the shoot comprising fresh media. For example , multiple shoot buds elongation /maintenance medium ( B002 ) is selected from developed in a bud induction medium ( B001 ) can be the group consisting of Nº- benzylaminopurine ( BAP ) , meta divided into individual pieces , and placed either on a fresh topolin (mT ) , zeatin , kinetin , 2 - isopentenyladenine ( 2ip ) , bud induction medium ( B001 ) , or on a fresh shoot elon gation /maintenance medium ( B002 ) ; multiple shoots adenine hemi sulfate , dimethylallyladenine, N-( 2 - chloro - 4 developed in a shoot elongation /maintenance medium pyridyl) -N ' -phenylurea ) ( 4 - CPPU ), and analogs of each ( BOO2 ) can be separated into individual pieces , and placed thereof. In some embodiments, the concentration of the one either on a fresh shoot elongation /maintenance medium or more cytokinins other than TDZ or an analog thereof is ( BOO2 ) , or a fresh bud induction medium ( B001 ) . Each from about 0.01 mg / L to about 100 mg/ L , e.g. , from about separated piece may comprise 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , or 0.25 mg / L to about 5 mg / L . In some embodiments, the bud more shoot buds or shoots . induction medium ( B001 ) and / or the shoot elongation / [ 0587 ] The present invention also provides methods for maintenance medium ( B002 ) further comprises one or plant micropropagation using a plant growth system more auxins , such as B -naphthoxyacetic acid (NAA ), 2,4 described herein . Dichlorophenoxy acetic acid ( 2,4 - D ) , indole - 3 -butyric acid [ 0588 ] In some embodiments, a plant growth system of the ( IBA ) , indole - 3 - acetic acid ( IAA ), picloram , and analogs of present invention is used for plant micropropagation . In each thereof. some embodiments, it is used for bamboo micropropagation . [ 0593 ] In some embodiments, the first incubation In some embodiments, it is used for micropropagation of sequence of 304 lasts for about half hour to about three Phyllostachys edulisi ‘Moso ', Phyllostachys bissetti, Farge weeks, e.g. , for about 24 hours to about 60 hours , and the sia denudata, Pleioblastus fortunei , Sasa Veitchii, Pleioblas second incubation sequence of 314 lasts for about 24 hours tus viridistriatus, Thamnocalamus crassinodus , Chusquea to about four weeks, e.g. , for about three days to about five Culeo " Cana Prieta " , Bambusa Old Hamii , Phyllostachys days. Moso, Phyllostachys Atrovaginata , Dendrocalamus Asper, [ 0594 ] In some embodiments, the length the plant propa or Guadua Angustifolia , Nigra Henon , Rufa, or Nigra . gation sequence is determined by the multiplication rate [ 0589 ] In some embodiments, a plant growth system of the reached . In some embodiments , the multiplication rate is present invention is used for plant micropropagation , from at least about 1,000 to at least about 100,000 within wherein the plant is a perennial, grass , or phtyo -pharmaceu about 3 weeks to about 6 months. tical plant. In some embodiments, it is used for micropropa [ 0595 ] As far as the inventors know , this is the first time gation of a perennial. The perennial can be an evergreen , that such a system has been used for plant micropropagation , deciduous, monocarpic, woody, or herbaceous perennial. In especially for bamboo micropropagation. The methods some embodiments, the perennial is Begonia , banana , gold using a bioreactor are unique at least for the following enrod , mint, agave , maple tree , pine tree, apple tree, alfalfa reasons : US 2020/0383331 A1 Dec. 10 , 2020 57
[ 0596 ] 1. The methods are suitable for both small scale Stage 2 medium . In some embodiments , the explants stay on ( e.g. , laboratory ) and large scale ( e.g. , industrial) plant Stage 2 medium for at least 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 or more micropropagation . rounds first before being transferred back to the Stage 1 [ 0597 ] 2. The methods allow the pulsing micropropaga medium . In some embodiments, the explants are on the tion technology described herein to be used in a more Stage 1 medium for about 1-36 hours ( e.g. , when spiked efficient way ( e.g. , recycled medium ; less labor; more accu media are used ) or more ( e.g. , when standard or reduced rate control; less contamination ; etc. ) . media are used ) followed by being transferred to the Stage [ 0598 ] 3. The methods enable greatly improved shoot / 2 medium . In some embodiments, the rotation is continuous plant multiplication over prior methods ( e.g. , micropropa until multiple shoots are observed . In some embodiments, gation using solid medium , and micropropagation using the rotation takes about 1 week to about 24 months , depend liquid medium without a bioreactor ). ing on plant species and media . [ 0599 ] 4. The methods enable greater plant survival rate [ 0605 ] Optionally, the multiplied shoots are then placed on over prior methods, particularly for certain plant species , a Stage 3 medium for further multiplication until desired such as Moso bamboo . number of shoots is obtained , depending on previous treat [ 0600 ] 5. Bamboo releases phenolics which are harmful to ments . The explants on the Stage 3 medium can be further the shoots / plants when they buildup in the media /environ transferred to a Stage 4 medium . In some embodiments, the ment, which has been a problem with using solid medium . explants are on the Stage 3 medium for about 1-36 hours [ 0601 ) Moving from solid growth environment ( e.g. , plant ( e.g. , when spiked media are used ) followed by being micropropagation in tissue culture tubes/ boxes) to the liquid transferred to the Stage 4 medium . In some embodiments, environment and combining pulsing methods and a biore the explants are on the Stage 3 medium for about 10-120 actor system , the present invention achieves a major days or more ( e.g. , when standard or reduced media are improvement in number of shoots /plants that are obtained , used ) followed by being transferred to the Stage 4 medium . as well as improving the resultant plants' health and ability In some embodiments , the explants remain the Stage 4 to produce full size plants. Without wishing to be bound by medium for about 10-120 days. any theory, the inventors believe these achievements are the [ 0606 ] Alternatively, the multiplied shoots obtained from result of controlling / reducing the exposure of the shoots / a Stage 2 medium can be rotated between at least one Stage plantlets to toxic components in the growth compositions 3 medium and at least one Stage 4 medium . In some ( e.g. , certain plant hormones , such as TDZ ) and / or plant embodiments , the rotation is continuous for at least 1 , 2 , 3 , produced by - products ( e.g., phenolics ), by utilizing the 4 , 5 , 6 , 7 , 8 , 9 , 10 or more cycles . In some embodiments, the bioreactor systems of the present invention . rotation is continuous until desired number of shoots . In [ 0602 ] In addition to the methods described above which some embodiments , the desired number of shoots is are based on using “ bud induction media (B001 ) ” and obtained by separation into new tubes and further expansion . “ shoot elongation /maintenance media (BOO2 ) ” combina In some embodiments , about one to ten shoots per tube are tion , the present invention also provides alternative plant obtained per multiplication cycle . micropropagation methods based on using “ Stage 1 media ” , [ 0607 ] In some embodiments, the explants are placed on a “ Stage 2 media ” , “ Stage 3 media ” , and / or more media . Stage 1 medium , a Stage 2 medium , and a Stage 3 medium [ 0603 ] In embodiments , the methods comprising using at in rotation , until desired number of shoots is obtained . In least one “ Stage 1 media ” and at least one " Stage 2 media ” , some embodiments, the multiplied shoots are then placed on and an explant. In some embodiments , the Stage 1 and Stage a Stage 4 medium . 2 media are used sequentially during plant propagation . In [ 0608 ] In some embodiments, the explants are placed on a some embodiments, the explants remain on the Stage 1 Stage 1 medium and Stage 2 medium in rotation , until medium for about 1 to about 36 hours ( e.g. , when spiked desired number of shoots is obtained . In some embodiments , media are used ) . In some embodiments, the explants remain the multiplied shoots are then placed on a rotation of a Stage on the Stage 1 medium for 10-120 days ( e.g. , when standard 3 medium and a Stage 4 medium . or reduced media are used ) . In some embodiments , the [ 0609 ] In some embodiments, the explants are placed on a explants stay on Stage 1 medium for at least 1 , 2 , 3 , 4 , 5 , 6 , Stage 1 medium for about 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , or more 7 , 8 , 9 , 10 or more rounds ( e.g. , in each round , explants are rounds, and then transferred to a Stage 2 medium for about transferred from an old Stage 1 medium to a fresh Stage 1 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , or more rounds , until desired medium ) before being transferred to the Stage 2 medium . In number of shoots is obtained . In some embodiments, the some embodiments, the explants remain on the Stage 2 multiplied shoots are then placed on a rotation of a Stage 3 medium for about 1 to about 36 hours ( e.g. , when spiked medium and a Stage 4 medium . media are used ) . In some embodiments, the explants remain [ 0610 ] In some embodiments, the explants are placed on a the Stage 2 medium for about 10-120 days ( e.g. , when Stage 1 medium first. In some embodiments, the explants are standard or reduced media are used ) . In some embodiments, on the Stage 1 medium for about 1-36 hours ( e.g. , when the explants stay on Stage 2 medium for at least 1 , 2 , 3 , 4 , spiked media are used ) . In some embodiments, the explants 5,6 , 7 , 8 , 9 , 10 or more rounds ( e.g. , in each round , explants are on the Stage 1 medium for about 10-120 days or more are transferred from an old Stage 2 medium to a fresh Stage ( e.g. , when standard or reduced media are used ) . In some 2 medium ) . embodiments, this step comprises 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 [ 0604 ] In some embodiments , the Stage 1 and Stage 2 or more rounds of fresh Stage 1 medium . Then , the explants media are used in rotation during plant propagation . In some are kept on a rotation of a Stage 2 medium and a Stage 3 embodiments , the rotation is continuous for at least 1 , 2 , 3 , medium , until desired number of shoots is obtained . In some 4 , 5 , 6 , 7 , 8 , 9 , 10 or more cycles . In some embodiments, the embodiments, the multiplied shoots are then placed on a explants stay on Stage 1 medium for at least 1 , 2 , 3 , 4 , 5 , 6 , Stage 4 medium . In some embodiments, the multiplied 7,8,9 , 10 or more rounds first before being transferred to the shoots remain on the Stage 4 medium for about 10-120 days . US 2020/0383331 A1 Dec. 10 , 2020 58
[ 0611 ] Still optionally , the multiplied shoots obtained from [ 0619 ] In some embodiments, the multiplication process a Stage 4 medium can be transferred onto a Stage 5 medium can continue substantially indefinitely by continuing to as described herein . In some embodiments , the shoots are separate and multiply shoots . In some embodiments, the placed on a Stage 5 medium for about 1-24 hours or more multiplication cycles can be repeated without initiating new ( e.g. , when spiked media are used ) . In some embodiments , explants for at least 1 month , at least 3 months, at least 6 the shoots are placed on a Stage 5 medium for about 10 to months, at least 12 months , at least 24 months, at least 36 120 days ( e.g. , when standard or reduced media are used ) . In months, or more. In some embodiments , the multiplication some embodiments , the shoots are transferred to small tissue cycles include 1-10 days per cycle , 2-9 days per cycle , 3-6 culturing boxes , such as the magenta boxes . days per cycle , 0.5-3 days per cycle , 4-5 days per cycle , [ 0612 ] Still optionally, the explants are kept on the Stage 0.5-1 day per cycle , 10-120 days per cycle , etc. 5 medium first until the desired number of shoots is [ 0620 ] The present invention has many advantages . With obtained , then transferred to a Stage 6 medium as described out wishing to be bound by any theory, the methods dis herein . In some embodiments, the shoots are placed on a closed herein do not require the use of seeds or inflorescence Stage 6 medium for about 1-24 hours or more ( e.g. , when to start plants , or selection of diseased starting plants, or the spiked media are used ) . In some embodiments, the shoots use of antibiotics , somatic embryogenesis, pseudospiklets, are placed on a Stage 6 medium for about 10 to 120 days or induction and / or reversion of flowering. For successful ( e.g. , when standard or reduced media are used ). growth following tissue culture, the produced plants do not [ 0613 ] In some embodiments, the explants obtained from require watering directly on the pot but remain robust with the Stage 4 medium are placed on a rotation of a Stage 5 overhead watering and do not require multiple adjustments medium and a Stage 6 media , until the desired number of to light intensity or humidity conditions prior to transfer to shoots is obtained . a greenhouse or other growing conditions . Moreover, media [ 0614 ] Still optionally , the explants kept on the Stage 6 can be free from polyaspartic acid ( s ) , seaweed concentrates medium are transferred to a Stage 7 medium as described and / or surfactants . These improvements over prior methods herein . In some embodiments , the shoots are placed on a provide even additional advantages related to the health of Stage 7 medium for about 1-24 hours or more ( e.g. , when produced plants and efficiency of growth and processing. spiked media are used ) . In some embodiments, the shoots [ 0621 ] In some embodiments, the present invention can be are placed on a Stage 7 medium for about 10 to 120 days used for grass propagation. In some embodiments, the ( e.g. , when standard or reduced media are used ). micropropagated plants have not been genetically modified . [ 0615 ] Still optionally, the multiplied shoots obtained Other particular embodiments exclude the use of timentin from a Stage 4 medium can be transferred onto a rotation of and / or kanamycin in the micropropagation procedure . a Stage 4 medium , a Stage 5 medium , and a Stage 6 medium [ 0622 ] In some embodiments, when the methods are used as described herein . for bamboo propagation , explants from a bamboo plant [ 0616 ] Still optionally, the multiplied shoots obtained between the age of 3 months and 3 years are used . In some from a Stage 7 medium can be transferred one or more other embodiments , a node from the cane with the lateral shoot additional media ( e.g. , a Stage 8 , a Stage 9 , etc.) for further just breaking the sheath can be used as the explants. In some propagation if needed. embodiments, each nodal section can be cut into 3-5 milli [ 0617 ] A variety of appropriate explants can be used in meter sections with the shoot intact . In some embodiments , accordance with the present disclosure . In certain embodi the outer sheaths can be peeled off and discarded and the ments according to the present disclosure , immature nodal remaining nodal section piece put into a 10 % bleach solution sections from stems can be used as the explant material. In with a final concentration of 0.6 % sodium hydrochloride. In one embodiment, the explants can be new growth canes with some embodiments, the explant in bleach solution can be the lateral shoots just breaking the sheath at nodal section ( s ) . placed onto a Lab Rotators , Adjustable speed , Barnstead / New growth canes include those obtained from the plant Lab line orbital Shaker (model number KS 260 ) shaker table within a current season or year , wherein such new growth for 1 hour at 6-9 revolutions per minute . The explants can canes can be obtained from any node on the plant. In one then be put into a 1 % bleach solution with a final concen particular embodiment, explant material includes or is lim tration of 0.06 % sodium hydrochloride, and be placed back ited to the third node from the base of a cane . onto the shaker table for 30 minutes . This 1 % bleach [ 0618 ] In some embodiments, the plant is a bamboo . solution step can then be repeated . Detailed methods for collecting and initially disinfecting [ 0623 ] In some embodiments, individual explants can then bamboo explants are described in WO / 2011 / 100762 , which be placed on a Stage 1 media ( 15-25 mL ) within a tube and is incorporated herein by reference in its entirety . In some the tubes can be placed into a regulated clean growth embodiments , the disinfectant such as dichloroisocyanuric chamber at a temperature of from 65 ° F. - 70 ° F. and a full acid , dichloroisanuric acid , trichlorotriazinetriona, mercuric spectrum light level of 36-54 umole /m² / s2. The initial Stage chloride, hydrogen peroxide , FungiGoneTM (bio World , Inc. , 1 media can be BO038 - iv at a pH of 5.7 . The explants can Dublin , Ohio ) , plant preservatives can be used . In some then be transferred to fresh BOO38 - iv media every 10-120 embodiments, following the initial disinfection , the outer days ( usually every 21 days ), with contaminated tubes being sheaths of a bamboo can be peeled off and discarded and the discarded . remaining piece can be put into an approximately 1 % to [ 0624 ] In some embodiments, if a spiked version of the about 50 % solution of a commercial bleach or a similar BO038 - iv media is utilized , the explants can be placed in disinfecting solution . In some embodiments , the bleach can the spiked media for 0.25 , 0.5 , 0.75 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , be heated to about 20-60 ° C. , such as 23-50 ° C. In some 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , embodiments, sonication and vacuum infiltration of the 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , tissue can also be used with the described disinfection 42 , 43 , 44 , 45 , 46 , 47 or 48 hours before transition to a procedures. " standard ” media disclosed herein or to a media containing US 2020/0383331 A1 Dec. 10 , 2020 59 substantially reduced or no cytokinins ( “ reduced ” media as Media BOO35 used herein ) for the remainder of the 10-120 day cycle . Additional time periods for placement in a spiked media [ 0626 ] include anywhere between 0.1 and 240 hours and can include , without limitation , 0.1-0.5 hours , 0.3-2.5 hours, Reduced Reduced Reduced Reduced Reduced 2.5-6 hours, 1-10 hours , 5-15 hours , 10-20 hours, 15-25 Component BOO35 - i BOO35 - ii BOO35-11i BOO35 - iv BOO35 - v hours, 20-30 hours , 25-35 hours , 30-40 hours , 35-45 hours, NAA 0.15 0.01 0.1 0.15 0.1 40-50 hours , 45-55 hours, 50-60 hours , 55-65 hours , 60-70 BAP 1 0.75 0.9 1 0.5 Thidiazuron 0.2 0.25 0.2 0.25 0.25 hours, 65-75 hours , 70-80 hours , 75-85 hours, 80-90 hours, Meta 4 . 5 4.5 5 5 85-95 hours , 90-100 hours, 95-105 hours , 100-110 hours , Topolin 105-115 hours , 110-120 hours , 115-125 hours , 120-130 hours , 125-135 hours, 130-140 hours, 135-145 hours , 140 150 hours, 145-155 hours , 150-160 hours , 155-165 hours , Media BO038 CPPU 160-170 hours, 165-175 hours , 170-180 hours , 175-185 hours, 180-190 hours , 185-195 hours, 190-200 hours , 195 [ 0627 ] 205 hours, 200-210 hours , 205-215 hours , 210-220 hours , 215-225 hours , 220-230 hours , 225-235 hours, 230-240 Reduced Reduced Reduced Reduced Reduced hours, 235-245 hours, 240-250 hours , 3-6 hours, 7-17 hours, BOO38 BO038 BOO38 BOO38 BOO38 12-22 hours , 17-27 hours , 22-32 hours, 27-37 hours , 32-42 Component CPPU - i CPPU - ii CPPU - iii CPPU - iv CPPU - v hours , 37-47 hours , 42-52 hours , 47-57 hours, 52-62 hours, NAA 0.05 0.04 0.025 0.04 0.05 57-67 hours , 62-72 hours, 67-77 hours , 72-82 hours , 77-87 BAP 1 1 0.5 0.9 1 hours, 82-92 hours, 87-97 hours, 92-102 hours , 97-107 Thidiazuron 0.2 0.3 0.75 0.7 0.5 Meta 3 3 5 4.5 2.5 hours , 102-112 hours , 107-117 hours, 112-122 hours , 117 Topolin 127 hours, 122-132 hours, 127-137 hours , 132-142 hours , CPPU 0.75 0.5 0.75 0.7 0.5 137-147 hours, 142-152 hours , 147-157 hours , 152-162 hours, 157-167 hours , 162-172 hours , 167-177 hours , 172 182 hours , 177-187 hours, 162-172 hours , 167-177 hours , Media BO038 DPU 182-192 hours , 187-197 hours , 192-202 hours , 197-207 hours, 202-212 hours , 207-217 hours, 212-222 hours , 217 [ 0628 ] 227 hours, 222-232 hours, 227-237 hours , 232-242 hours, 237-247 hours or 242-252 hours . Placement in spiked media Reduced Reduced Reduced Reduced Reduced can also be 0.5 hours less than a cycle in standard or reduced BO038 BO038 BO038 BOO38 BOO38 media , 1 hour less than a cycle in standard or reduced media Component CPU - i CPU - ii CPU - iii CPU - iv CPU - v and all time periods in between 1 and 240 hours less than a NAA 0.01 0.05 0.025 0.05 0.05 cycle in standard or reduced media . Alternatively, in place of BAP 0.25 1 0.75 1 0.5 Thidiazuron 0.2 0.6 0.75 0.75 0.3 spending the remainder of the cycle in the standard or Meta 2 3 4 . 5 reduced media, explants can be placed on a spiked media for Topolin a period of time followed by culture on a standard or reduced DPU 0.75 0.75 0.6 0.75 0.4 media for the full cycle time ( i.e. 10-120 days not reduced by time spent in the spiked media ). [ 0629 ] Alternatively, the cytokinins noted above can be [ 0625 ] Media containing no cytokinins or substantially replaced with weaker cytokinins at similar or higher levels . reduced cytokinins can be a reduced B0036 media , reduced Exemplary weaker cytokinins include zeatin and kinetin . BO040 media , reduced BO037 media , reduced BO031 [ 0630 ] Contaminated tubes can be identified by bacterial media , reduced BO038 media , reduced BOO28 media , discoloration of the agar or by visible surface contamination . reduced BOO29 media , reduced BOO30 media , reduced These explants can stay on the chosen B0038 - iv media for BOO39 media , reduced BO041 media , reduced BOO42 3-4 10-120 day cycles ( usually 21 day cycles ) or as modified media , reduced BOO43 media , reduced BOO44 media , in the spiked procedure ( spiked media for a period of 0.25 , reduced BOO35 media , reduced BOO38 CPPU media , 0.5 , 0.75 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , reduced BO038 DPU media with all cytokinins and / or 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , auxins removed or can have at least one cytokinin and / or 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 or auxin's amount reduced by 1 % , 5 % , 10 % , 15 % , 20 % , 25 % , 48 hours before transition to a standard or reduced media for 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , the remainder of the 10-120 day cycle or for a full 10-120 80 % , 85 % , 90 % , 95 % , 98 % , 99 % , 100 % , 1-10 % , 5-15 % , day cycle ) . Additional time periods for placement in a spiked 10-20 % , 15-25 % , 20-30 % , 25-35 % , 30-40 % , 35-45 % , media include anywhere between 0.1 and 240 hours and can 40-50 % , 45-55 % , 50-60 % , 55-65 % , 60-70 % , 65-75 % , include, without limitation , 0.1-0.5 hours , 0.3-2.5 hours , 70-80 % , 75-85 % , 80-90 % , 85-95 % , 90-100 % , 3-6 % , 2.5-6 hours, 1-10 hours , 5-15 hours, 10-20 hours , 15-25 7-17 % , 12-22 % , 17-27 % , 22-32 % , 27-37 % , 32-42 % , hours , 20-30 hours, 25-35 hours , 30-40 hours, 35-45 hours, 37-47 % , 42-52 % , 47-57 % , 52-62 % , 57-67 % , 62-72 % , 40-50 hours , 45-55 hours , 50-60 hours , 55-65 hours , 60-70 67-77 % , 72-82 % , 77-87 % , 82-92 % or 87-97 % . Non - limit hours , 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, ing examples of reduced media include ( embodiments with 85-95 hours, 90-100 hours , 95-105 hours , 100-110 hours, no cytokinins or auxins not shown in table format ): 105-115 hours, 110-120 hours , 115-125 hours , 120-130 US 2020/0383331 A1 Dec. 10 , 2020 60 hours, 125-135 hours , 130-140 hours , 135-145 hours , 140 2.5-6 hours, 1-10 hours , 5-15 hours, 10-20 hours , 15-25 150 hours, 145-155 hours, 150-160 hours , 155-165 hours , hours , 20-30 hours, 25-35 hours, 30-40 hours, 35-45 hours, 160-170 hours, 165-175 hours , 170-180 hours, 175-185 40-50 hours, 45-55 hours , 50-60 hours , 55-65 hours , 60-70 hours, 180-190 hours , 185-195 hours, 190-200 hours , 195 hours , 65-75 hours, 70-80 hours, 75-85 hours, 80-90 hours, 205 hours, 200-210 hours, 205-215 hours , 210-220 hours , 85-95 hours, 90-100 hours , 95-105 hours, 100-110 hours, 215-225 hours, 220-230 hours , 225-235 hours , 230-240 105-115 hours, 110-120 hours , 115-125 hours , 120-130 hours, 235-245 hours , 240-250 hours , 3-6 hours, 7-17 hours , hours , 125-135 hours , 130-140 hours, 135-145 hours , 140 12-22 hours , 17-27 hours, 22-32 hours, 27-37 hours , 32-42 150 hours , 145-155 hours, 150-160 hours , 155-165 hours, hours, 37-47 hours , 42-52 hours , 47-57 hours, 52-62 hours , 160-170 hours , 165-175 hours , 170-180 hours , 175-185 57-67 hours , 62-72 hours , 67-77 hours , 72-82 hours , 77-87 hours , 180-190 hours , 185-195 hours, 190-200 hours , 195 hours , 82-92 hours, 87-97 hours , 92-102 hours , 97-107 205 hours , 200-210 hours, 205-215 hours, 210-220 hours, hours, 102-112 hours , 107-117 hours, 112-122 hours, 117 215-225 hours , 220-230 hours, 225-235 hours , 230-240 127 hours , 122-132 hours , 127-137 hours , 132-142 hours , hours , 235-245 hours, 240-250 hours, 3-6 hours , 7-17 hours, Izgl47 hours, 142-15 hours , 147-15 hours, 152-16 12-22 hours, 17-27 hours , 22-32 hours, 27-37 hours , 32-42 hours, 157-167 hours, 162-172 hours, 167-177 hours , 172 hours , 37-47 hours, 42-52 hours , 47-57 hours, 52-62 hours, 182 hours , 177-187 hours, 162-172 hours , 167-177 hours, 57-67 hours, 62-72 hours , 67-77 hours, 72-82 hours , 77-87 182-192 hours , 187-197 hours , 192-202 hours , 197-207 hours , 82-92 hours, 87-97 hours , 92-102 hours, 97-107 hours , 202-212 hours , 207-217 hours, 212-222 hours , 217 hours , 102-112 hours, 107-117 hours, 112-122 hours , 117 227 hours, 222-232 hours , 227-237 hours , 232-242 hours , 127 hours , 122-132 hours , 127-137 hours , 132-142 hours , 237-247 hours or 242-252 hours. Placement in spiked media 137-147 hours , 142-152 hours , 147-157 hours, 152-162 can also be 0.5hours less than acyclein standardor reduced hours , 157-167 hours , 162-172 hours, 167-177 hours , 172 media , 1 hour less than a cycle in standard or reduced media 182 hours , 177-187 hours, 162-172 hours, 167-177 hours, and all time periods in between 1 and 240 hours less than a 182-192 hours , 187-197 hours , 192-202 hours , 197-207 cycle in standard or reduced media . If an explant is cultured hours , 202-212 hours , 207-217 hours , 212-222 hours , 217 on a particular spiked media type ( e.g. BO038 ) , when 227 hours , 222-232 hours, 227-237 hours , 232-242 hours, transferred to a standard or reduced media , the standard or 237-247 hours or 242-252 hours. Placement in spiked media reduced media can be of the same type ( e.g. standard or can also be 0.5 hours less than a cycle in standard or reduced reduced BO038 ) or of a different type ( e.g. standard or media , 1 hour less than a cycle in standard or reduced media reduced BO039 , BO040 , BOO44 , BOO30 , BOO36 etc. ). and all time periods in between 1 and 240 hours less than a [ 0631 ] In particular embodiments disclosed herein , culture cycle in standard or reduced media . periods are less than 12 weeks, less than 9 weeks , or less [ 0636 ] Generally, one - ten shoots per tube can be obtained than 6 weeks. per multiplication cycle . [ ] Explants can be taken offthemedia after the third [ 0637 ] Following removal from the multiplication pro cycle if multiplication is occurring. If multiplication is not cess , the shoots can be transferred to small tissue culturing occurring or not occurring to a significant degree, explants boxes (known as “ magenta boxes ” ) for 10-120 days ( usually can be left on the media for a fourth cycle 14-21 days) containing a Stage 3 , Stage 4 or Stage 5 media , [ 0633 ] Live shoots can next be transferred to a Stage 2 in this Example, BO046 at a pH of 5.7 for 10-120 days media ( if standard BO038 used in the previous step or a ( usually 14-21 days ) or B0047 at a pH of 5.7 for 10-120 Stage 3 media if a basic spiked procedure was used ), such days ( usually 14-21 days ) . As above , shoots can be placed in as BOO36 , BOO39 , BO040 , BOO41 , BOO42 , BOO43 , spiked media for shorter time periods followed by placement BO044 , BOO30 , BOO35 , BOO38 CPPU or BO038 DPU at into a standard or reduced media for the remainder of or for a pH of 5.7 . The cultures can stay on this Stage 2 media until a full 10-120 day cycle . the desired number of shoots is obtained by separation into [ 0638 ] In particular embodiments disclosed herein , culture new tubes and further expansion . Generally, the range of periods are less than 12 weeks , less than 9 weeks , or less time includes 10-120 day cycles ( usually 14-21 day cycles ) than 6 weeks. between which the cultures are assigned to go through [ 0639 ] As will be understood by one of ordinary skill in another multiplication round or transitioned to a Stage 3 or the art, when spiked media are used , the use of the spiked Stage 4 media, for example , BO037 - iv or BO031 - iv at a pH media increases the number of media stages within a par of 5.7 for further multiplication . ticular process due the following use of a standard or [ QG34 Inparticularembodiments disclosedherein , culture reduced media . If spiked media are used at only one stage , periods are less than 12 weeks, less than 9 weeks , or less the process generally expands by 1 media stage . If spiked than 6 weeks . media are used at two stages , the process generally expands [ 0635 ] Alternatively, live shoots can also be placed on a by 2 media stages . If spiked media are used at three stages , spiked BOO36 , BO039 , B0040 , B0041 , B0042 , B0043 , the process generally expands by 3 media stages , etc. BO044 , BOO30 , BO035 , BO038 CPPU , BOO38 DPU [ 0640 ] Alternatively, the following procedures may also media for a period of 0.25 , 0.5 , 0.75 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , be used ( Stage 1 , Stage 2 , Stage 3 , etc , media are defined IQ , 13,14,15,16,17,18,19,20,2l , 22,23,24,25 , elsewhere herein ): 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , [ 0641 ] Individual explants can then be placed on a Stage 42 , 43 , 44 , 45 , 46 , 47 or 48 hours before transition to a same 1 media ( 15-25 mL ) within a tube and the tubes placed into or different type of standard or reduced media for the a regulated clean growth chamber at a temperature of from remainder of the 10-120 day cycle or for a full 10-120 day 65 ° F. - 70 ° F. and a full spectrum light level of 36-90 cycle . Additional time periods for placement in a spiked umole/ mº / s². The Stage 1 media can be standard BOO38 - iv media include anywhere between 0.1 and 240 hours and can at a pH of 5.7 or spiked BO038 - iv media . If placed on include , without limitation , 0.1-0.5 hours , 0.3-2.5 hours, standard BO038 - iv , the explants can be transferred to fresh